Activating mitotic checkpoint control mechanisms

ABSTRACT

Provided herein are compositions and methods for the treatment of cancer by activating the spindle assembly checkpoint (SAC) in cells. In particular, dimerized Mps1 and Spc105/KNL1 constructs are provided as tunable activators of SAC, allowing for control of chromosome segregation accuracy and prevention of aneuploidies that are common in cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

The present invention is a continuation of U.S. patent application Ser. No. 16/836,335, filed on Mar. 31, 2020, which is a continuation of U.S. patent application Ser. No. 15/355,824, filed Nov. 18, 2016, now U.S. Pat. No. 10,669,320, which claims the priority benefit of U.S. Provisional Patent Application 62/256,971, filed Nov. 18, 2015, each of which is incorporated by reference in its entirety.

STATEMENT REGARDING FEDERAL FUNDING

This invention was made with government support under grants GM105948 and GM112992 awarded by the National Institutes of Health. The government has certain rights in the invention.

SEQUENCE LISTING

The text of the computer readable sequence listing filed herewith, titled “34587-304_SEQUENCE_LISTING”, created Feb. 24, 2023, having a file size of 30,308 bytes, is hereby incorporated by reference in its entirety.

FIELD

Provided herein are compositions and methods for the treatment of cancer by activating the spindle assembly checkpoint (SAC) in cells. In particular, dimerized Mps1 and Spc105/KNL1 constructs are provided as tunable activators of SAC, allowing for control of chromosome segregation accuracy and prevention of aneuploidies that are common in cancer.

BACKGROUND

Genetic instability, which includes both numerical and structural chromosomal abnormalities, is a hallmark of cancer. The spindle assembly checkpoint (SAC) is a mechanism by which cells ensure proper chromosome segregation and thereby maintain the euploid status of cells. Breakdown of the SAC contributes to cellular aneuploidy, which can lead to tumorigenesis and cancer. Compositions that prevent aneuploidy-related cancers are needed.

SUMMARY

Provided herein are compositions and methods for the treatment of cancer by activating the spindle assembly checkpoint (SAC) in cells. In particular, dimerized Mps1 and Spc105/KNL1 constructs are provided as tunable activators of SAC, allowing for control of chromosome segregation accuracy and prevention of aneuploidies that are common in cancer.

In some embodiments, provided herein are systems comprising: (a) an Mps1 polypeptide linked to a first dimerization element; and (b) a Spc105/KNL1 polypeptide linked to a second dimerization element, wherein dimerization of the first dimerization element and second dimerization element facilitates phosphorylation of the Spc105/KNL1 polypeptide by the Mps1 polypeptide. In some embodiments, the Mps1 polypeptide comprises a kinase domain having at least 70% sequence similarity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or ranges therebetween) with all or a portion of a kinase domain of a wild-type Mps1 protein (SEQ ID NO:2), and retains all or a portion of the kinase activity of the wild-type Mps1 protein. In some embodiments, the Mps1 polypeptide comprises a kinase domain having at least 70% sequence identity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or ranges therebetween) with all or a portion of a kinase domain of a wild-type Mps1 protein (SEQ ID NO:2), and retains all or a portion of the kinase activity of the wild-type Mps1 protein. In some embodiments, the Mps1 polypeptide comprises at least 70% sequence identity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or ranges therebetween) with a portion a wild-type Mps1 protein (e.g., comprising a kinase domain) of at least 50 amino acids (e.g. >50 amino acids, >100 amino acids, >150 amino acids, >200 amino acids, >250 amino acids, >300 amino acids, >350 amino acids). In some embodiments, the Mps1 polypeptide comprises at least 70% sequence identity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or ranges therebetween) with a portion a wild-type Mps1 protein of not more than 500 amino acids (e.g. <500 amino acids, <450 amino acids, <400 amino acids, <350 amino acids, <300 amino acids, <250 amino acids, <200 amino acids, <150 amino acids, <100 amino acids). In some embodiments, the Spc105/KNL1 polypeptide comprises a phosphodomain having at least 70% sequence similarity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or ranges therebetween) with all or a portion of a phosphodomain of a wild-type Spc105 (SEQ ID NO:8) or KNL1 (SEQ ID NO:5) protein, and retains all or a portion of the capacity of the wild-type Spc105 or KNL1 protein to be phosphorylated by Mps. In some embodiments, the Spc105/KNL1 polypeptide comprises a phosphodomain having at least 70% sequence identity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or ranges therebetween) with all or a portion of a phosphodomain of a wild-type Spc105 (SEQ ID NO:8) or KNL1 (SEQ ID NO:5) protein, and retains all or a portion of the capacity of the wild-type Spc105 or KNL1 protein to be phosphorylated by Mps. In some embodiments, the Spc105/KNL1 polypeptide comprises at least 70% sequence identity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or ranges therebetween) with a portion of a wild-type Spc105 (SEQ ID NO:8) or KNL1 (SEQ ID NO:5) protein of at least 50 amino acids (e.g. >50 amino acids, >100 amino acids, >150 amino acids, >200 amino acids, >250 amino acids, >300 amino acids, >350 amino acids, >400 amino acids, >450 amino acids, >500 amino acids). In some embodiments, the Spc105/KNL1 polypeptide comprises at least 70% sequence identity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or ranges therebetween) with a portion of a wild-type Spc105 (SEQ ID NO:8) or KNL1 (SEQ ID NO:5) protein of not more than 500 amino acids (e.g. <500 amino acids, <450 amino acids, <400 amino acids, <350 amino acids, <300 amino acids, <250 amino acids, <200 amino acids, <150 amino acids, <100 amino acids). In some embodiments, the phosphorylation of the Spc105/KNL1 polypeptide by the Mps1 polypeptide is sufficient to activate a spindle assembly checkpoint (SAC) is a cell within which the phosphorylation occurs. In some embodiments, the first or second dimerization element is Frb and the other dimerization element is Fkbp12. In some embodiments, systems further comprise a dimerization inducer, wherein the dimerization inducer tunably alters the degree of dimerization in a concentration dependent manner.

In some embodiments, provided herein are compositions comprising an Mps1 polypeptide linked to a dimerization element. In some embodiments, the Mps1 polypeptide comprises a kinase domain having at least 70% sequence similarity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or ranges therebetween) with all or a portion of a kinase domain of a wild-type Mps1 protein (SEQ ID NO:2), and retains all or a portion of the kinase activity of the wild-type Mps1 protein. In some embodiments, the Mps1 polypeptide comprises a kinase domain having at least 70% sequence identity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or ranges therebetween) with all or a portion of a kinase domain of a wild-type Mps1 protein (SEQ ID NO:2), and retains all or a portion of the kinase activity of the wild-type Mps1 protein. In some embodiments, the dimerization element is Frb or Fkbp12.

In some embodiments, polypeptides and constrcuts are modified or provided in delivery systems to reduce immunogenicity.

In some embodiments, provided herein are compositions comprising a Spc105/KNL1 polypeptide linked to a dimerization element. In some embodiments, the Spc105/KNL1 polypeptide comprises a phosphodomain having at least 70% sequence similarity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or ranges therebetween) with all or a portion of a phosphodomain of a wild-type Spc105 (SEQ ID NO:8) or KNL1 (SEQ ID NO:5) protein, and retains all or a portion of the capacity of the wild-type Spc105 or KNL1 protein to be phosphorylated by Mps. In some embodiments, the Spc105/KNL1 polypeptide comprises a phosphodomain having at least 70% sequence identity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or ranges therebetween) with all or a portion of a phosphodomain of a wild-type Spc105 or KNL1 protein, and retains all or a portion of the capacity of the wild-type Spc105 (SEQ ID NO:8) or KNL1 (SEQ ID NO:5) protein to be phosphorylated by Mps. In some embodiments, the dimerization element is Frb or Fkbp12.

In some embodiments, provided herein are methods of activating a spindle assembly checkpoint (SAC) in a cell comprising administering to the cell a system or composition comprising: a Spc105/KNL1 polypeptide linked to a dimerization element and/or a Mps1 polypeptide linked to a dimerization element. In some embodiments, activating the SAC prevents aneuploidies in the cell. In some embodiments, the cell is within a tissue, organ, or subject, and the SAC is activated in all or a group of cells within the tissue, organ, or subject. In some embodiments, activating the SAC treats or prevents cancer in the cells, tissue, organ, or subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-E. (a) The steps in the kinetochore-based signaling cascade of the SAC (P's indicate Mps1-mediated phosphorylation) that may be disrupted by microtubule attachment. (b) Top: Protein architecture of the metaphase kinetochore-microtubule attachment. Bottom: Schematic of the rapamycin-induced dimerization technique used to anchor Mps1 to the C terminus of Mtw1 (Mtw1-C). (c) Top: Micrographs show the anchoring of Mps1-Frb-GFP at Mtw1-C (time after rapamycin addition indicated; scale bars, ˜3 μm). The stereotypical distribution of kinetochores in between the spindle pole bodies in metaphase visualized with Mtw1-GFP and Spc97-mCherry is shown on the right. Schematic underneath depicts the metaphase spindle morphology. Bottom: Kinetics of rapamycin-induced anchoring of Mps1-Frb-GFP to Mtw1-C. (d) Left: Representative transmitted-light images of yeast cells before and 1 h after the addition of rapamycin to anchor Mps1 at Mtw1-C. Right: Localization of Bub1-GFP and Mad1-GFP, and kinetochores (visualized by Spc24-mCherry) in untreated cells (control) and in cells that have Mps1 anchored at Mtw1-C(+RAP). Scale bar, ˜3 μm. (e) Top. Domain organization of Spc105. The end-to-end length of the unstructured domain of Spc105 (amino acids 1-455) is predicted to be 11.7±5 nm (mean±s.d using the worm-like chain model). The maximum length of its α-helical region (amino acids 455-709) is 38 nm (3.6 amino acids per turn with a 0.54 nm pitch). The predicted kinetochore-binding domain is ˜6 nm long. The depiction is not drawn to scale. The six Mps1 phosphorylation sites are depicted as bars. Bottom: Cell-cycle progression of asynchronous cells with the indicated genotypes observed on anchoring Mps1 at Mtw1-C. Accumulation of large-budded cells indicates mitotic arrest.

FIG. 2A-F. Effects of anchoring key SAC regulators to Mtw1-C on the cell cycle. (a) Top: Representative images display the expected localization of SAC proteins tagged with Frb-GFP in untreated cells and one hour after the addition of rapamycin. Bottom: Benomyl sensitivity of indicated strains. (b) Representative transmitted light micrographs of four strains treated with rapamycin for 135 minutes to anchor Mps1, Ipl1, Mad1, or Glc7, at Mtw1-C. The bar graph displays the percentage of large-budded in each case averaged from two independent experiments. (c) Effect of the ATP analog 1-NAPP1 on the localization of the Ipl1 substrate Sli15-GFP in cells expressing ipl1-as6, an analog-sensitive allele of the Ipl1 kinase. Representative pre-anaphase cells expressing Sli15-GFP are shown on the right. Quantification of Sli15-GFP fluorescence on the shown on the left. Spindle localization of Sli15-GFP significantly increased following 1-NAPP1 treatment indicating that the analog inhibits ipl1-as6. (d) Cell cycle kinetics following the release of S-phase synchronized cells into media containing 1-NAPP1 and rapamycin. Blocking ipl1-as6 activity did not have any effect on SAC activation induced by Mps1 anchored at Mtw1-C. (e) Bar graph: Frequency of prometaphase and metaphase cells with kinetochore-localized Mps1. Spindle length was used to classify cells as prometaphase or metaphase cells. Scatter plot (mean±95% confidence interval; n=21, 46 and 66 kinetochore clusters from left to right) displays the abundance of kinetochore-localized Mps1-Frb-GFP in prometaphase, metaphase-arrested cells (by repressing CDC20), and when it is anchored to Mtw1-C in heterozygous diploid strains. (f) Quantification of Mps1 localization to kinetochores soon after release from metaphase compared to that in. Micrographs on the right show localization of Mps1 relative to spindle pole bodies over a period of 6 minutes during the metaphase to anaphase transition.

FIG. 3A-C. Testing the sensitivity of SAC signaling steps to the attachment status of the kinetochore. (a) Cell-cycle progression of three different strains following release from metaphase. Solid lines indicate cell-cycle progress of a strain expressing Mtw1-2xFkbp12 and Mps1-Frb released into media with or without rapamycin. The dotted line indicates cell-cycle progression of a mad2Δ strain similarly released from metaphase arrest. Plotted points represent the average values calculated from independent experiments. (b) Separation between the centroids of fluorescently labelled kinetochore proteins along the spindle axis obtained by high-resolution co-localization in unperturbed metaphase cells (Ctrl) and rapamycin-treated cells (Rap-rapamycin added to anchor Mps1 at Mtw1-C. (c) Left: Fractional intensity distributions of Mps1-Frb-GFP (which autonomously localizes along the spindle in the absence of rapamycin) and Ndc80-GFP along the spindle in cells arrested in metaphase using CDC20 repression (spindle pole bodies visualized using Spc97-mCherry). Right: Bub3 and Mad1 do not localize to kinetochores under the same conditions. Mad1 puncta correspond to its known localization to the nuclear envelope. Scale bar, ˜3 μm.

FIG. 4A-D. The ability of Mps1 to activate the SAC depends on its position in the kinetochore. (a) Fluorescence recovery after photobleaching of Mps1-frb-GFP anchored at Ndc80-C(circles), and loss of anchored protein from the unbleached cluster (squares). Dashed line shows the expected rate of photobleaching as a result of imaging determined in cells expressing Ndc80-GFP. Scale bar, ˜3 μm. (b) Top: Structure of the Ndc80 complex and the positions of fluorescent tags used for FRET. Scatter plot: Proximity ratio, which is directly proportional to the FRET efficiency35, for FRET between Spc25-mCherry or Nuf2-mCherry and Mad1-Frb-GFP anchored to Spc24-C. The proximity ratio is defined as the acceptor fluorescence resulting from FRET normalized by the sum of mCherry cross-excitation and GFP emission bleed-through into the mCherry imaging channel. FRET between the anchored donor, Mad1-Frb-GFP, and the acceptor, Spc25-mCherry, was readily detected, but it was absent when the mCherry was fused to Nuf2-C. Spc25-C is <3 nm away from Spc24-C, where the donor is anchored, whereas Nuf2-C is >10 nm away. Mad1 was used, rather than Mps1, in this experiment to ensure that the number of donors is equal to the number of acceptor molecules (either Spc25-mCherry or Nuf2-mCherry) for accurate FRET quantification. (c) Number of protein molecules anchored at Ndc80-C, measured 30 min after rapamycin addition. (d) Top: The organization of yeast kinetochore proteins along the microtubule axis. The N-terminal half of Spc105 is not drawn to scale. Bottom: Bar graph shows the number of colonies formed on rapamycin-containing plates relative to control plates. Right: Representative photographs of plates for three strains.

FIG. 5A-C. Kinase activity of the kinetochore-anchored Mps1 is sufficient for SAC activation. (a) Cells expressing the analog-sensitive Mps1 allele, mps1-as1 or wild type Mps1 were treated as indicated at the top. Bar graph displays the percentage of two-budded cells (which form when a mitotic cell fails to sustain the SAC in the presence of a damaged spindle and produce a new bud by re-entering the cell cycle). (b) Inhibition of the diffusible mps1-as1 does not affect SAC activation by Mps1-Frb anchored at the kinetochore. Heterozygous diploid strains expressing mps1-as1 and Mps1-Frb were synchronized in S-phase and released into 1-NMPP1 for 15 minutes. Rapamycin was then added to anchor Mps1-Frb at Mtw1-C. The anchored Mps1 arrested the cell cycle robustly, and the cells retained the large-budded morphology for a prolonged period of time. (c) Micrographs: Mad1 localization relative to the spindle pole body in haploid cells that have Mps1 anchored to the indicated subunit. Bar graph displays the percentage of cells with visible Mad1 localization in between the spindle poles in each case. The corresponding metaphase spindle length in each case is presented in the scatter plot.

FIG. 6A-G. Cell cycle effects of anchoring Mps1, Ipl1 or Mad1 constitutively within the kinetochore. (a) Cell cycle kinetics of asynchronous cultures where Mps1 is anchored at the C-termini of indicated kinetochore subunits in wild-type or SAC null strains (mad2A). (b) Quantification of Mps1-Frb-GFP anchored at indicated kinetochore subunits measured 45 minutes after rapamycin treatment and normalized relative to endogenous Mps1 in metaphase-arrested cells. The recruitment of Mps1 at Dad4-C and Ctf19-C that activates the SAC is comparable to that at Ask1-C which does not activate the SAC. (c) Reducing the length of the flexible linker connecting Mps1 and Frb (from 24 to 7 amino acids) did not change the effect of anchoring Mps1 on colony growth. (d-e) The number of colonies formed on rapamycin-containing plates relative to control plates, when Ipl1 (in d) or Mad1 (in e) is constitutively anchored at the indicated positions. (f) Mad1 anchored at N-Ndc80 generates unattached kinetochores (arrowheads) in a large fraction of cels. (g) Graph presents the fraction of cells expressing Spc105-6A or Spc105 that arrested with large-buds when Mad1 was anchored at N-Ndc80 (rapamycin treatment for 4 hours).

FIG. 7A-F. The Dam1 complex defines a boundary for SAC signaling by anchored Mps1. (a) Schematic: Position of the Dam1 complex relative to the Ndc80 complex23 and subunit organization within the Dam1 complex. EMD1372 was used to infer the dimensions of the Dam1 complex. (b) Colony growth (also see Supplementary FIG. 4 a ) on control (Ctrl) and rapamycin (+Rap) plates. The number of days after plating is indicated at the top; the anchoring subunit is indicated on the left. (c) Cell-cycle progression when Mps1 is anchored to a Dam1 subunit (indicated on the left) in cells released from an experimentally imposed S-phase arrest. S-phase synchronization was used to ensure that the kinetochores formed end-on attachments and loaded the Dam1 complex before Mps1 was anchored. (d) Normalized distribution of Dad4-mCherry on the spindle when Mps1 was anchored to the indicated positions for 1 h. Control data are from untreated metaphase cells. Micrographs on the right show the localization of Dad4-mCherry relative to that of Mps1-frb-gfp anchored to the indicated subunits (scale bars, ˜3 μm). (e) The separation between kinetochore clusters in the cells in d, measured as the separation between maximum-intensity pixels in the two Dad4-mCherry puncta in each cell. Although there is a small decrease in kinetochore cluster separation when Mps1 is anchored at Dad3-C, cell-cycle progression is unaffected as seen in c. (f) Left: Classification of Dam1 complex subunits inferred from the Mps1 anchoring experiments. Right: Activity map of the anchored Mps1 along the length of the kinetochore-microtubule attachment. Arrows from the Dam1 complex depict the proposed orientation of the C termini of subunits used as anchors.

FIG. 8A-B. Anchoring Mps1 to Dam1 subunits leads to different phenotypes. (a) Subunit organization of the Dam1 complex, and bar graph displaying the number of colonies formed on rapamycin relative to the control plates. The total numbers of colonies scored are displayed at the bottom. (b) Cell cycle kinetics of rapamycin treated (to anchor Mps1 at indicated subunits) or untreated (control) cells.

FIG. 9A-E. Phosphorylation of the Spc105 phosphodomain by Mps1 is sufficient to activate the SAC. (a) Schematic of Spc105¹²⁰⁻³²⁹, the minimal Spc105 phosphodomain. NLS: nuclear localization signal used to send Spc105^(120:329) to the nucleus. (b) Cell-cycle kinetics following rapamycin addition to anchor the phosphorylatable (solid black line) or non-phosphorylatable Spc105^(120:329) (solid grey line) to Mps1-C. The dashed black line shows the cell-cycle progression of the mad2Δ strain after anchoring Spc105^(120:329) to Mps1. (c) Localization of Spc105^(120:329) or Spc105^(120: 329:6A) when anchored to Mps1. Scale bars, ˜3 μm. (d) Strategy to anchor Spc105^(120:329) at N-Ndc80, and the localization of Spc105^(120:329) at the indicated times after rapamycin addition. Scale bar, ˜3 μm. (e) Recruitment of Mad1 to the kinetochore clusters when Spc105^(120:329) (top) or Spc105^(120: 329:6A) (bottom) is anchored at NNdc80. Asterisk: known Mad1 localization at the nuclear envelope. Scale bar, ˜3 μm.

FIG. 10A-B. SAC signaling induced by rapamycin-induced dimerization of Spc105^(120:329) and Mps1 does not require functional kinetochores. (a) Representative images show Spc105^(120:329) anchored to Mps1 (rapamycin treatment for 45 minutes) localizing to the kinetochores. Mad1 also co-localizes with these kinetochore clusters. (b) Cells carrying the temperature-sensitive ndc10-1 allele and expressing Spc105^(120:329) and Mps1-Fkbp12 were treated as indicated at the top. When released at the restrictive temperature from G1 arrest, these cells go through the cell cycle without assembling functional kinetochores and fail in cytokinesis, and give rise to cells with two buds (black bars; also see transmitted light micrograph top-right). However, when the same experiment was conducted in rapamycin containing media, the emergence of two budded cells was delayed by an hour (light gray bars).

FIG. 11 . SAC signaling induced by Mps1 anchored at N-Ndc80 depends on the attachment-state of the kinetochore. S-phase synchronized cells were treated as indicated in the schematic at the top and the percentage of large-budded cells formed after 100 minutes was measured as an indicator of cell cycle arrest Mps1 anchored at Mtw1-C constitutively activated the SAC in the presence of attachments and in nocodazole. However, Mps1 anchored at N-Ndc80 allowed normal cell cycle progression and caused cell cycle arrest only in the presence of unattached kinetochores in nocodazole.

FIG. 12A-E. Spc105^(120:329) activates the SAC only when it is anchored in the outer kinetochore. (a) Representative micrographs of asynchronously dividing cells showing the localization of Spc105^(120:329) and cell-cycle progression as a function of the anchoring position (indicated at the top; scale bar, ˜3 μm). Large-budded cells with <2 μm separation between kinetochore clusters were characterized as metaphase-arrested cells. (b) Accumulation of metaphase-arrested cells after rapamycin addition, when either Spc105^(120:329) (solid lines) or its non-phosphorylatable version, Spc105^(120: 329:6A) (dashed lines), was anchored at the indicated positions. (c) Mad1-mCherry localization after anchoring Spc105^(120:329) at the indicated positions for 1 h (scale bar, ˜3 μm). The bar graph shows the fraction of metaphase cells that recruit Mad1 to the kinetochores in each case. Total number of cells analysed in each case is indicated on top of the bars. (d) Top: Cell-cycle progression as in a when a modified version of the Spc105 phosphodomain that includes the Glc7-recruitment motif (Spc105^(120:329), solid lines) or its non-phosphorylatable version (Spc105^(120: 329:6A) dashed line) was anchored at the indicated kinetochore positions. Bottom: Micrographs (scale bar, ˜3 μm) and quantification of kinetochore-localized Bub3-mCherry 45 min after either Spc105^(120:329) or Spc105^(120: 329:6A) was anchored at Ask1-C in cells arrested in metaphase using CDC20 repression. (e) Map of the SAC activity of the anchored Spc105^(120: 329).

FIG. 13A-B. The proximity between the CH domains of Ndc80 and the phosphodomain of Spc105 within the kinetochore controls SAC signaling. (a) Scatter plot: Proximity ratio measurements for FRET between mCherry-Nuf2 or mCherry-Ndc80 and GFP-Spc105 in attached (metaphase) and unattached (nocodazole-treated) kinetochores. (b) Cell-cycle kinetics after anchoring Spc105^(120: 329) at the indicated positions in strains expressing spc105^(-6A).

FIG. 14A-D. Effect of spindle disruption on SAC protein recruitment and kinetochore architecture. (a) Spindle disruption with nocodazole generates two or three kinetochore clusters within the nuclei of most budding yeast cells as reported previously. The cluster that contained majority of the kinetochores (large, asterisks) localized proximal to the collapsed spindle pole bodies (visualized by Spc97-GFP). One or two smaller kinetochore clusters (small, arrowheads) were found distal to the spindle pole bodies. Bar graph displays the percentage of large or small clusters that are proximal to the spindle pole body. (b) The smaller kinetochore clusters (arrowheads) in nocodazole recruit significantly higher levels of Mps1 and Bub1 than the large cluster. (c) Dam1 complex (visualized with Ask1-mCherry) is retained at the SAC-inactive cluster, whereas it is significantly reduced at the SAC-active clusters in nocodazole. Quantification of Ask1-mCherry fluorescence measured relative to Spc24-mCherry fluorescence is displayed on the right. (d) Measurement of FRET between GFP-Spc105 and either mCherry-Nuf2 or mCherry-Ndc80 in SAC-active and SAC-inactive kinetochore clusters. FRET between mCherry-Nuf2 (or mCherry-Ndc80) and GFP-Spc105 in the SAC-inactive kinetochore cluster is higher than metaphase FRET value, and significantly lower than the FRET observed in the SAC-active cluster.

FIG. 15 . Spc105^(120: 329:6A) restores the SAC when it is anchored to unattached kinetochores in SAC-null strains. Top: Experimental scheme. Bar graph: Fraction of nocodazole-treated cells with two buds in the presence and absence of rapamycin in cells expressing spc105-6A (see micrographs on the left). When Spc105^(120: 329:6A) was anchored either at N-Ndc80 or at Spc24-C, it restored the SAC. The cells arrested with large buds (transmitted light micrograph on the right). In this condition, Spc105^(120: 329:6A) is visible as multiple puncta corresponding to kinetochore clusters that form when budding yeast cells are treated with nocodazole.

FIG. 16 . Induced dimerization of KNL-1 phosphodomain (M3-M3) & Mps1 induces a prolonged metaphase arrest. Top: representative micrographs. Bottom left: Mps1 is expressed at equivalent levels in the analyzed cells. Bottom right: Time spent in metaphase (0 min: begin observation).

FIG. 17A-E. Cytosolic dimerization of the Mps1 kinase domain and a minimal KNL1 phosphodomain is sufficient to induce metaphase arrest. (A) Diagram of the SAC signaling cascade. Black arrows imply protein recruitment to the kinetochore, gray arrows mark cytoplasmic reactions. (B) Exemplary scheme for conditional dimerization Mps1 with the minimal KNL1 phosphodomain. (C) Top, Bright-field and fluorescence images of HeLa cells from time lapse imaging display the indicated proteins. Elapsed time (minutes) indicated in the top left corner. Scale bar ˜2.4 microns. Bottom, Duration of mitosis in a 2-hour time-lapse experiment (n=55 and 98 for DMSO and Rapamycin respectively). (D) Time until anaphase entry after treatment with either DMSO (n=30) or Reversine (n=27, ≥2 independent trials) of cells in rapamycin-induced arrest. (E) Top, eSAC schematic. Left, Duration of mitosis for untreated and rapamycin-treated cells (n=629 and 2705 respectively). Top right, Psuedocolored fluorescence image of a cell in rapamycin-induced metaphase arrest stained as indicated, Bottom right: absence of kinetochore localization of the phosphodomain visualized by neon fluorescence (scale bar ˜1.2 μm). Horizontal lines indicate mean±s.e.m. intervals in all scatterplots.

FIG. 18A-F. eSAC induces metaphase arrest in a kinetochore-independent manner. (A) Phosphoregulation of the eSAC phosphodomain and KNL1 analyzed by immunoblotting for the indicated proteins. (B) Left, Reversine treatment inactivates the SAC and significantly accelerates cell division (n=432 and 199 for untreated and Reversine-treated cells respectively, Mann-Whitney test). Right, Effect of Reversine on eSAC activity based on the partially Reversine-resistant Mps1S611R kinase domain (n=621 and 1193 for Rapamycin and Rapamycin+Reversine respectively; 2 trials). (C) Effect of the Aurora B inhibitor ZM447439 on the eSAC-induced mitotic arrest (n=931 and 205 for Rapamycin and Rapamycin+ZM447439 respectively; 2 trials). (D) Activity of the membrane-targeted eSAC (n=697 and 1056 for untreated and Rapamycin-treated cells respectively, 2 trials). Right, Confocal images display protein localizations as indicated. Scale bar ˜5 μm. (E) Mass spectrometry analysis of immunoprecipitated eSAC phosphodomain under the indicated conditions. (F) Effect of RNAi-mediated depletion of either BubR1 or Mad2 on eSAC activity (n=78, 390, 300,191, 72 and 140 respectively, 2 trials). Bar height=mean, error bars display s.e.m. Horizontal lines in scatter plots display mean+/−s.e.m.

FIG. 19A-I. Dose-response characteristics of the eSAC reveal mechanisms that achieve ultra-high sensitivity and automatic gain modulation. (A) Hypothetical relationships between the number of signaling kinetochores and SAC signal. (B) Schematics of the eSAC phosphodomains. c, Montages of bright-field images and fluorescence heat-maps of representative cells (Δt=20 minutes for 1-MELT and 30 minutes for 6-MELT phosphodomain montage). (D-F) Dose (Mps1 kinase domain fluorescence at the beginning of mitosis) vs. response (duration of mitosis) relationship for the indicated eSAC phosphodomains (n=2572, 2791, and 2705 respectively from >2 trials). Duration of mitosis at 0 eSAC abundance was obtained from the mean duration of mitosis for the respective cell line in the absence of rapamycin. Each gray circle represents one cell. Open squares represent the means of binned data; error bars represent s.e.m. Curves in d and e: 4-parameter sigmoid fits. Curve in D: Lowess filtered data. (G) Dose-response curves for all five phosphodomains. (H) Activation thresholds and (I) maximal mitotic duration calculated from either the sigmoidal fits or graphically by interpolation for F.

FIG. 20A-C. Mathematical model of the eSAC and simulation of the dose-response curves. (A-B) Model of eSAC phosphodomains containing 1 and 4 MELT repeats respectively. It represents the binding of all SAC proteins by the single “Bub” unit. The amino acid sequence of each MELT repeat (labeled by its repeat number in KNL1) determines its binding affinity for Bub. Graphs display the steady-state abundance of different Bub-bound phosphodomain species (left) and the rate of closed/active Mad2 formation stimulated by these phosphodomains (right) as a function of eSAC activator concentration. Each species is designated by the MELT repeat numbers that it contains, the specific MELT bound by Bub. (C) Simulated dose-response curves obtained by relaying the signaling activity of the eSAC model to a previously described model for a bistable switch that governs mitotic exit. The dashed and solid black curves display the dose-response characteristics for the phosphodomain with 6 MELT repeats without and with synergistic activity respectively.

FIG. 21A-B. Assessment of kinetochore-localization domains within KNL1. (A) Fluorescent images showing the localization of the indicated KNL1 regions as GFP fusions in live HeLa cells (numbers alongside each micrograph denote the residues in each construct). The kinetochore-binding domain at the C-terminus of KNL1 strongly localized to kinetochores. Residues proximal to the N-terminus also transiently localized to kinetochores only in prometaphase (Scale bar=5 μm). (B) Images at the top display metaphase cells with misaligned chromosomes. KNL1 fragments containing the N-terminus also localized to kinetochores on these chromosomes. Dashed boxes highlight the magnified areas shown in the bottom right panel.

FIG. 22A-B. Schematic of eSAC cell-line construction. (A) Each eSAC cell line was created by integrating a bi-cistronic cassette in the HeLa genome at a unique Loxp site via Cre-mediated recombination. (B) The Phosphodomain-neonGFP-2xFkbp12, wherein the phosphodomain cassette can contain a specified numbers of MELT repeats, is constitutively expressed by the EF1α promoter. The 5′ UTR of this gene also contains a sequence encoding shRNA against the endogenous FKBP protein. Frb-mCherry-Mps1, wherein Mps1 can be either the full length gene or just the sequence encoding its kinase domain is expressed conditionally from a TetON promoter.

FIG. 23A-B. Phosphorylation of the MELT repeats in the minimal KNL1 phosphodomain by the Mps1 kinase domain is necessary for eSAC activity. (A) Kinase activity of the Mps1 kinase domain is necessary for the rapamycin-induced mitotic arrest. Rapamycin-induced dimerization of the analog-sensitive allele of the Mps1 kinase domain, Mps1S602A, with the minimal phosphodomain (displayed in the cartoon at the top) produced a weak mitotic arrest. The weak eSAC-induced arrest likely indicates a significantly reduced activity of the mutant kinase domain. Combined treatment with Rapamycin and the ATP analog 1NM-PP1 (10 μM) abrogated the eSAC-induced arrest. Cells expressing >5 a. u. of Frb-mCherry-Mps1S602A were used for this analysis (n=177, 206, and 332 respectively; p<0.0001, Mann-Whitney test). (B) Phosphorylatable MELT repeats are necessary for rapamycin-induced mitotic arrest. The minimal phosphodomain used in the experiment is displayed at the top. n=853, 2238, 252, and 313 respectively. In both A and B, black horizontal lines display mean±s.e.m.

FIG. 24A-B. Duration of the eSAC-induced metaphase arrest correlates strongly with the cellular abundance of the kinase domain, but not with the abundance of the phosphodomain. (A-B) Dependence of eSAC-induced mitotic duration on the abundance of the phosphodomain (neonGreen fluorescence) and Mps1 kinase domain (mCherry fluorescence) shown for minimal KNL1 phosphodomains containing 2 and 6 MELT repeats respectively (n=1376 for a and n=1877 for b). The surface was calculated using the ‘griddata’ function with cubic interpolation in MatLab.

FIG. 25A-B. The abundance of the eSAC kinase domain is comparable with that of the endogenous Mps1 kinase. (A) Comparison of the expression level of the endogenous Mps1 and the eSAC Mps1 kinase domain (Frb-mCherry-Mps1⁵⁰⁰⁻⁸⁵⁷) for three different cell lines expressing the indicated phosphodomains. Total cell lysates probed with an antibody against the C-terminus of Mps1, which is present in the kinase domain used for the eSAC. (B) Assessment of the expression levels of the eSAC phosphodomains (antibody against the Fkbp12 protein) and their phosphorylation after rapamycin-induced dimerization with Mps1 (phosphospecific antibody against MELT¹³ does not recognize the phosphodomain containing MELT¹² alone).

FIG. 26A-E. Dose-response characteristics for the indicated phosphodomains (n=2572, 2969, 3043, 2791, and 2705 respectively from N≥2 independent trials). Open squares represent mean values of binned data, error bars represent s.e.m.

FIG. 27A-C. The eSAC dose-response relationship is maintained even when it is localized to the plasma membrane, and when the endogenous Mps1 kinase is inhibited. (A-B) Dose-response characteristics of a phosphodomain containing 6 MELT repeats when it is cytosolic (a, n=2705 re-plotted from FIG. 19F) and when it is targeted to the plasma membrane (b, n=1056). (C) The complex nature of the dose-response characteristics is retained even when the endogenous SAC activation switch is inactivated by inhibiting the kinase activity of the endogenous Mps1. Open squares and circles represent averages of binned data, error bars represent s.e.m.

FIG. 28A-E. Abundance of different Bub-bound species for the five eSAC phosphodomains at different concentrations of the eSAC activator complexes. (A-E) The abbreviations for different Bub-bound phosphodomain species are as follows. The subscripted number following the M denotes the rank of the MELT repeat in the KNL1 phosphodomain (see FIG. 4A). For example, M₁₂ symbolizes the eSAC phosphodomain with one MELT motif, and M_(12,13,14_12,13,14) symbolizes the phosphodomain with six MELT repeats. The subscript ‘B’ in front of the number signifies that the MELT motif denoted by the number is bound by Bub. The concentration of Bub is assumed to be 30 a.u.

FIG. 29 . Schematic of the model used to simulate anaphase onset. An active eSAC produces the Closed form of Mad2, which sequesters Cdc20 as part of the MCC. This eSAC activity is promoted by high CyclinB-CDK1, and inhibited by an unspecified phosphatase (CAPP). High CyclinB-CDK1 activity also inhibits the activity of the phosphatase. eSAC catalyzes the formation of the closed/active Mad2 at a rate k_(amad) determined by: (a) the amino acid sequences of the MELT repeats in the eSAC phosphodomain, (b) potentially synergistic activity of the MELT repeats, and (c) abundance of the eSAC activator. Closed/active Mad2 sequesters Cdc20, and thus inhibits the APC. Free Cdc20 acts with APC to degrade Cyclin B and to promote the dissociation of the Mad2-Cdc20 complex. Thus, k_(amad) also determines the rate of degradation of Cyclin B by the APC. When Cyclin B levels fall below the minimum threshold value, the two feedback loops work concurrently to rapidly drive the cell out of mitosis.

FIG. 30A-D. (A) Dependence of the weighted average rate of generation of Closed/active Mad2, k_(amad′) by different eSAC phosphodomain plotted on the eSAC abundance. (B) k_(amad) curves for eSAC phosphodomains containing 2 and 6 MELT repeats, along with their corresponding bifurcation curves (k_(amad) values above/below which the switch is ON/OFF). (C) Time dependence of [CycB] for different phosphodomains. It is assumed that the cell exits mitosis when the Cyclin B concentration falls below 5 a.u. (dashed line). (D) Dependence of time in mitosis on [eSAC] for different phosphodomains. The dashed curve corresponds to dose response curve for the phosphodomain containing 6 MELT repeats calculated by assuming that cooperativity is absent.

DEFINITIONS

As used herein “CASC5” and “KNL1” refer to the same human protein (Pubmed AccessionNo.Q8NG31) that is involved in spindle-assembly checkpoint signaling, correct chromosome alignment during mitosis, and attachment of the kinetochores to the spindle microtubules. “Spc105” refers to analogous protein in yeast (Pubmed Accession No.P53148). “Spc105/KNL1” is used herein to refer collectively to these proteins and other variants thereof.

As used herein, the term “aneuploidy” refers to an abnormal number of chromosomes within a cell. Aneuploidy includes an imbalance of genetic material caused by loss or gain of part of any chromosome (segmental aneuploidy). Accordingly, in some embodiments, aneuploid cells may have three copies of part of one chromosome and only one copy of part of the other chromosome. In other embodiments, aneuploid cells may contain an addition or deletion of one or more entire (whole) chromosomes. In other embodiments, aneuploid cells may contain an addition or deletion of one or more chromosomal arms or portions thereof. Accordingly, in some embodiments, aneuploid cells may be monosomic, trisomic, tetrasomic, etc., for one or more chromosomes or chromosomal regions. In some embodiments, aneuploid cells may have a loss of one or both copies of one or more chromosomes or chromosomal regions. In some embodiments, a region of about 0.01%, about 0.1%, about 1%, about 10%, about 25%, about 50%, or a higher, lower, or intermediate percentage of each of one or more chromosomes may be lost (e.g., one copy or both copies absent from a cell) or duplicated (e.g., three, four, or more copies in a cell). It should be appreciated that aneuploidy also may be associated with one or more additional chromosomal rearrangements including translocations, inversions, etc., of one or more chromosomal regions.

As used herein, the terms “Mps1 element”, “Mps1 component”, and “Mps1 polypeptide” refer to a polypeptide that is capable of performing the Spc105/KNL1-phosphorylating function of Mps-1 and activating the SAC. In some embodiments, an Mps1 element or polypeptide comprises significant sequence identity (e.g., >70%) with wild-type Mps1 (SEQ ID NO:1) or a fragment thereof (e.g., all or a portion of the Mps-1 kinase domain (SEQ ID NO:2)). In some embodiments, phosphorylation of Spc105/KNL1 or a Spc105/KNL1 polypeptide by an Mps-1 polypeptide is sufficient for activating the SAC.

As used herein, the terms “Spc105/KNL1 element”, “Spc105/KNL1 component”, and “Mps1 element polypeptide”, refer to polypeptide that is capable of being phosphorylated by Mps1 (or an Mps1 polypeptide) and activating the SAC. In some embodiments, an Spc105/KNL1 polypeptide comprises significant sequence identity (e.g., >70%) with wild-type Spc105 (SEQ ID NO:7) or KNL1 (SEQ ID NO:4) or a fragment thereof (e.g., all or a portion of the phosphodomain of Spc105 (SEQ ID NO: 8) or KNL1 (SEQ ID NO: 5)). In some embodiments, phosphorylation of an Spc105/KNL1 polypeptide by Mps1 or an Mps1 polypeptide is sufficient for activating the SAC.

As used herein, the term “tunable” refers to the adjustability of an activity within a system. A particular activity may be adjustable by controlling the level or concentration of one or more components responsible for the activity, or by the inclusion of an enhancer or inhibitor of the activity or an interaction responsible for the activity. For example, the activity of a complex (e.g., dimer) may be “tuned” by altering (e.g., increasing or decreasing) the concentration of one or more components (e.g., Spc105/KNL1 and Mps1 fragments) of the complex, and/or by the altering the concentration (or the presence or absence) of one or more effectors (e.g., enhancer, inhibitor, etc.) of complex (e.g., dimer) formation.

As used herein, the term “dimer” refers to a noncovalent complex of two protein, polypeptide, and/or peptide components. In some embodiments, a first protein, polypeptide, and/or peptide component comprises a dimerization domain (e.g., a peptide or polypeptide segment) to facilitate dimerization of a functional domain with a second protein, polypeptide, and/or peptide component. In some embodiments, a first protein, polypeptide, and/or peptide component is linked to a non-peptide/non-polypeptide element to facilitate dimerization with a second protein, polypeptide, and/or peptide component.

As used herein, unless otherwise specified, the terms “peptide” and “polypeptide” refer to polymer compounds of two or more amino acids joined through the main chain by peptide amide bonds (—C(O)NH—). The term “peptide” typically refers to short amino acid polymers (e.g., chains having fewer than 25 amino acids), whereas the term “polypeptide” typically refers to longer amino acid polymers (e.g., chains having more than 25 amino acids).

As used herein, the term “phosphodomain” refers to a portion of a protein, polypeptide, or peptide that is the substrate for a kinase and is phosphorylated thereby under appropriate conditions. A phosphodomain is sufficient to support phosphorylation outside of the context of a greater protein sequence. The term “phosphorylation site” refers to a position or group of several amino acids within a phosphodomain where phosphorylation occurs. The phosphorylation site may not be capable of supporting phosphorylation outside of the context of a phosphodomain. A phosphodomain may comprise multiple phosphorylation sites. A protein or polypeptide may comprise one or more phosphodomains.

As used herein, the term “wild-type,” refers to a gene or gene product (e.g., protein) that has the characteristics (e.g., sequence) of that gene or gene product isolated from a naturally occurring source, and is most frequently observed in a population. In contrast, the term “mutant” refers to a gene or gene product that displays modifications in sequence when compared to the wild-type gene or gene product. It is noted that “naturally-occurring mutants” are genes or gene products that occur in nature, but have altered sequences when compared to the wild-type gene or gene product; they are not the most commonly occurring sequence. “Synthetic mutants” are genes or gene products that have altered sequences when compared to the wild-type gene or gene product and do not occur in nature. Mutant genes or gene products may be naturally occurring sequences that are present in nature, but not the most common variant of the gene or gene product, or “synthetic,” produced by human or experimental intervention.

A “conservative” amino acid substitution refers to the substitution of an amino acid in a polypeptide with another amino acid having similar properties, such as size or charge. In certain embodiments, a polypeptide comprising a conservative amino acid substitution maintains at least one activity of the unsubstituted polypeptide. A conservative amino acid substitution may encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include, but are not limited to, peptidomimetics and other reversed or inverted forms of amino acid moieties. Naturally occurring residues may be divided into classes based on common side chain properties, for example: hydrophobic: norleucine, Met, Ala, Val, Leu, and Ile; neutral hydrophilic: Cys, Ser, Thr, Asn, and Gln; acidic: Asp and Glu; basic: His, Lys, and Arg; residues that influence chain orientation: Gly and Pro; and aromatic: Trp, Tyr, and Phe. Non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class; whereas conservative substitutions may involve the exchange of a member of one of these classes for another member of that same class.

As used herein, the term “percent sequence identity” refers to the degree (e.g., 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, etc.) to which two polymer sequences (e.g., peptide, polypeptide, nucleic acid, etc.) have the same sequential composition of monomer subunits. If two polymers have identical sequences (e.g., 100% sequence identity) they may be referred to herein as having “sequence identity.” The term “percent sequence similarity” refers to the degree (e.g., 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, etc.) with which two polymer sequences (e.g., peptide, polypeptide, nucleic acid, etc.) have similar polymer sequences. For example, similar amino acids are those that share the same biophysical characteristics and can be grouped into the families (see above). If two polymers have sequences that have monomers at each position that share the same biophysical characteristics they may be referred to herein as having “sequence similarity.” The “percent sequence identity” (or “percent sequence similarity”) is calculated by: (1) comparing two optimally aligned sequences over a window of comparison (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window, etc.), (2) determining the number of positions containing identical (or similar) monomers (e.g., same amino acids occurs in both sequences, similar amino acid occurs in both sequences) to yield the number of matched positions, (3) dividing the number of matched positions by the total number of positions in the comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window), and (4) multiplying the result by 100 to yield the percent sequence identity or percent sequence similarity. For example, if peptides A and B are both 20 amino acids in length and have identical amino acids at all but 1 position, then peptide A and peptide B have 95% sequence identity. If the amino acids at the non-identical position shared the same biophysical characteristics (e.g., both were acidic), then peptide A and peptide B would have 100% sequence similarity. As another example, if peptide C is 20 amino acids in length and peptide D is 15 amino acids in length, and 14 out of 15 amino acids in peptide D are identical to those of a portion of peptide C, then peptides C and D have 70% sequence identity, but peptide D has 93.3% sequence identity to an optimal comparison window of peptide C. For the purpose of calculating “percent sequence identity” (or “percent sequence similarity”) herein, any gaps in aligned sequences are treated as mismatches at that position.

As used herein, the term “pharmaceutically acceptable carrier” refers to non-toxic solid, semisolid, or liquid filler, diluent, encapsulating material, formulation auxiliary, or carrier conventional in the art for use with a therapeutic agent for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. The pharmaceutically acceptable carrier is appropriate for the formulation employed. For example, if the therapeutic agent is to be administered orally, the carrier may be a gel capsule. If the therapeutic agent is to be administered subcutaneously, the carrier ideally is not irritable to the skin and does not cause injection site reaction.

As used herein, the term “effective amount” refers to the amount of a composition (e.g., pharmaceutical composition) sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.

As used herein, the term “administration” refers to the act of giving a drug, prodrug, or other agent, or therapeutic treatment (e.g., pharmaceutical compositions of the present invention) to a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs. Exemplary routes of administration to the human body can be through the eyes (e.g., intraocularly, intravitrealy, periocularly, ophthalmic, etc.), mouth (oral), skin (transdermal), nose (nasal), lungs (inhalant), oral mucosa (buccal), ear, rectal, by injection (e.g., intravenously, subcutaneously, intratumorally, intraperitoneally, etc.) and the like.

As used herein, the terms “co-administration” and “co-administer” refer to the administration of at least two agent(s) (e.g., first and second SAC-activating dimerization constructs, SAC-activating dimerization constructs and dimerization inducer, system described herein and second cancer therapy, etc.) or therapies to a subject. In some embodiments, the co-administration of two or more agents or therapies is concurrent (e.g., in the same or separate formulations). In other embodiments, a first agent/therapy is administered prior to a second agent/therapy. Those of skill in the art understand that the formulations and/or routes of administration of the various agents or therapies used may vary. The appropriate dosage for co-administration can be readily determined by one skilled in the art. In some embodiments, when agents or therapies are co-administered, the respective agents or therapies are administered at lower dosages than appropriate for their administration alone. Thus, co-administration is especially desirable in embodiments where the co-administration of the agents or therapies lowers the requisite dosage of a potentially harmful (e.g., toxic) agent(s).

As used herein, the term “vector” refers to a polynucleotide that is used to express a polypeptide of interest in a host cell. A vector may include one or more of the following elements: an origin of replication, one or more regulatory sequences (such as, for example, promoters and/or enhancers) that regulate the expression of the polypeptide of interest, and/or one or more selectable marker genes (such as, for example, antibiotic resistance genes and genes that can be used in colorimetric assays, e.g., β-galactosidase). One skilled in the art can select suitable vector elements for the particular host cell and application at hand.

As used herein, the terms “treat,” “treatment,” and “treating” refer to reducing the amount or severity of a particular condition (e.g., angiogenesis), disease state (e.g., cancer), or symptoms thereof, in a subject presently experiencing or afflicted with the condition or disease state. The terms do not necessarily indicate complete treatment (e.g., total elimination of the condition, disease, or symptoms thereof). “Treatment,” encompasses any administration or application of a therapeutic or technique for a disease (e.g., in a mammal, including a human), and includes inhibiting the disease, arresting its development, relieving the disease, causing regression, or restoring or repairing a lost, missing, or defective function; or stimulating an inefficient process. Treatment may be achieved with surgery, radiation, and/or administration of one or more molecules, including, but not limited to, small molecules and polymers, such as polypeptides.

As used herein, the terms “prevent,” “prevention,” and preventing” refer to reducing the likelihood of a particular condition or disease state (e.g., cancer) from occurring in a subject not presently experiencing or afflicted with the condition or disease state. The terms do not necessarily indicate complete or absolute prevention. For example “preventing cancer” refers to reducing the likelihood of cancer occurring in a subject not presently experiencing or diagnosed with cancer. In order to “prevent cancer” a composition or method need only reduce the likelihood of cancer, not completely block any possibility thereof. “Prevention,” encompasses any administration or application of a therapeutic or technique to reduce the likelihood of a disease developing (e.g., in a mammal, including a human).

As used herein, the term “gene therapy” refers to the transfer of genetic material (e.g., DNA or RNA) of interest into a host to treat or prevent a genetic or acquired disease or condition. The genetic material of interest encodes a product, the production of which is desired in vivo. For a review see, in general, the text “Gene Therapy” (Advances in Pharmacology 40, Academic Press, 1997; herein incorporated by reference in its entirety).

DETAILED DESCRIPTION

Provided herein are compositions and methods for the treatment of cancer by activating the spindle assembly checkpoint (SAC) in cells. In particular, dimerized Mps1 and Spc105/KNL constructs are provided as tunable activators of SAC, allowing for control of chromosome segregation accuracy and prevention of aneuploidies that are common in cancer.

Experiments were conducted during development of embodiments herein that demonstrate that the microtubule-dependent proximity of two kinetochore proteins acts like a mechanical switch that controls SAC signaling. It also explains a functional significance of the stereotypical ‘end-on’ kinetochore-microtubule attachment and the nanoscale protein organization within this attachment.

The SAC is a surveillance mechanism that detects kinetochores that are not attached to the cell division apparatus. Even if one kinetochore is unattached, the SAC must arrest cell division to allow it to attach. However, the ability of the SAC to arrest the cell and its timely silencing depend on the expression levels of nine signaling proteins (refs.17, 18; herein incorporated by reference in their entireties) that participate in a cascade of five reactions (refs.17,19-23; herein incorporated by reference in their entireties). Aberrant expression of one or more proteins, which is common in tumor cells, and which occurs during aging, likely changes SAC signaling properties and leads to chromosome missegregation and genomic instability.

The proteins that form the mechanical switch for the SAC, Mps1, Hec1/Ndc80, and Spc105/KNL1, are all aberrantly expressed in cancer cells, and therefore, important potential targets of therapeutics. KNL1 (a.k.a. CASC5) is the human version and Spc105 is the yeast version of a protein encoded by this gene is a component of the multiprotein assembly that is required for creation of kinetochore-microtubule attachments and chromosome segregation. Analysis of the yeast kinetochore in both budding yeast and in HeLa cells during development of embodiments herein demonstrates that the SAC can be activated by inducing the dimerization of Mps1 and the phosphodomain of Spc105/KNL1 in the cytosol. This finding demonstrates that the ‘wait-anaphase’ signal can be generated to activate the SAC in a kinetochore-independent fashion.

In some embodiments, provided herein is a tunable SAC activator. In some embodiments, the SAC activator is genetically encoded. In some embodiments, the SAC activators herein provide control of the SAC signal in both cell lines and in whole animals (e.g., animal models, humans, patients, etc.). In some embodiments, the level of control is titratable. In some embodiments, compositions, methods, and systems are provided for the tunable activation of SAC and temporary cell cycle arrest (e.g., in metaphase) to allow for proper chromosome segregation, cell division, to prevent aneuploidy, and/or to treat or prevent cancer.

In some embodiments, the tunable SAC activators herein comprise an Mps1 component (e.g., a fragment of Mps-1, or a variant thereof, capable of phosphorylating Spc105/KNL1) and a Spc105/KNL1 component (e.g., a fragment of Spc105/KNL1, or a variant thereof, comprising one or more phosphodomains and capable of being phosphorylated by Mps1). In some embodiments, a tunable SAC activator comprises dimerizable Mps1 and Spc105/KNL1 components. In some embodiments, upon dimerization, the Mps1 component phosphorylates one or more phosphodomains on the Spc105/KNL1 component, thereby initiating/facilitating/enhancing the SAC cascade and temporary cell cycle arrest.

In some embodiments, a first component (e.g., Mps1 element) of a tunable SAC activator comprises an Mps1 domain (e.g. kinase domain). In some embodiments, the Mps1 domain is the functional domain of the first component of the tunable SAC activator. In some embodiments, the first component further comprises a dimerization domain (e.g., a peptide or polypeptide segment that facilitates dimerization with a second component of the tunable SAC activator) or a dimerization element (e.g., a non-peptide/non-polypeptide element that facilitates dimerization with a second component of the tunable SAC activator. In some embodiments, a second component (e.g., Spc105/KNL1 element) of a tunable SAC activator comprises an Spc105/KNL1 domain (e.g., phosphodomain). In some embodiments, the Spc105/KNL1 domain is the functional domain of the second component of the tunable SAC activator. In some embodiments, the second component further comprises a dimerization domain (e.g., a peptide or polypeptide segment that facilitates dimerization with a first component of the tunable SAC activator) or a dimerization element (e.g., a non-peptide/non-polypeptide element that facilitates dimerization with a first component of the tunable SAC activator. In some embodiments, the dimerization domain and/or dimerization element on the first component facilitates dimerization of the first and second components via noncovalent interaction with a dimerization domain and/or dimerization element on the second component.

In some embodiments, the Mps1 domain of the Mps1 element comprises the full-length sequence of Mps-1 (e.g., a naturally-occurring sequence). In some embodiments, the Mps1 domain of the Mps1 element comprises a synthetic variant of full-length Mps1 (e.g., comprising one or more non-naturally-occurring conservative or non-conservative substitutions relative to the wild-type Mps1). In some embodiments, the Mps1 domain of the Mps1 element comprises a fragment of Mps1. In some embodiments, the Mps1 domain of the Mps1 element comprises the Mps1 kinase domain. In some embodiments, the Mps1 domain of the Mps1 element comprises a fragment of Mps1 with one or more non-naturally-occurring substitutions (e.g., conservative or non-conservative substitutions). In embodiments in which the Mps1 domain of the Mps1 element comprises a synthetic variant and/or fragment of Mps1, the Mps1 domain is capable of phosphorylating one or more phosphodomains of Spc105/KNL1, or a variant or fragment (e.g., upon dimerization of the Mps1 element with a second element comprising a Spc105/KNL1 domain). In some embodiments, the Mps1 domain comprises at least 50% sequence identity (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or any ranges there between) with full length Mps1 (SEQ ID NO:1) or a fragment thereof. In some embodiments, the Mps1 domain comprises at least 50% sequence similarity (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or any ranges there between) with full length Mps1 (SEQ ID NO: 1) or a fragment thereof. In some embodiments, the Mps1 domain comprises only conservative substitutions with respect to full length Mps1 (SEQ ID NO:1). In some embodiments, the Mps1 domain comprises at least 50% sequence identity (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or any ranges there between) with the kinase domain of Mps1 (SEQ ID NO:2) or a fragment thereof. In some embodiments, the Mps1 domain comprises at least 50% sequence similarity (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or any ranges there between) with the kinase domain of Mps1 (SEQ ID NO:2) or a fragment thereof. In some embodiments, the Mps1 domain comprises only conservative substitutions with respect to the kinase domain of Mps1 (SEQ ID NO:2) or a fragment thereof.

In some embodiments, the Spc105/KNL1 domain of the Spc105/KNL1 element comprises the full-length sequence of Spc105/KNL1 (e.g., a naturally-occurring Spc105 or KNL1 sequence). In some embodiments, the Spc105/KNL1 domain of the Spc105/KNL1 element comprises a synthetic variant of full-length Spc105/KNL1 (e.g., comprising one or more non-naturally-occurring conservative or non-conservative substitutions relative to a naturally-occurring Spc105 or KNL1 sequence). In some embodiments, the Spc105/KNL1 domain of the Spc105/KNL1 element comprises a fragment of a Spc105 or KNL1 sequence. In some embodiments, the Spc105/KNL1 domain of the Spc105/KNL1 element comprises a fragment of Spc105 or KNL1 with one or more non-naturally-occurring substitutions (e.g., conservative or non-conservative substitutions). In embodiments in which the Spc105/KNL1 domain of the Spc105/KNL1 element comprises a synthetic variant and/or fragment of Spc105 or KNL1, the Spc105/KNL1 domain is capable of being phosphorylated at one or more phosphodomains by Mps1 or an active fragment and/or variant thereof (e.g., upon dimerization of the Spc105/KNL1 element with a second element comprising a Mps1 domain). In some embodiments, the Spc105/KNL1 domain comprises at least 50% sequence identity (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or any ranges there between) with a full-length naturally-occurring Spc105 (SEQ ID NO:7) or KNL1 (SEQ ID NO:4) or a fragment thereof. In some embodiments, the Spc105/KNL1 domain comprises at least 50% sequence similarity (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or any ranges there between) with full length Spc105 (SEQ ID NO:7 or KNL1 (SEQ ID NO:4) or a fragment thereof. In some embodiments, the Spc105/KNL1 domain comprises only conservative substitutions with respect to full length Spc105 (SEQ ID NO:7 or KNL1 (SEQ ID NO:4) (SEQ ID NO:1). In some embodiments, the Spc105/KNL1 domain comprises at least 50% sequence identity (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or any ranges there between) with the phosphodomain of Spc105 (SEQ ID NO:8) or KNL1 (SEQ ID NO:5) or a fragment thereof. In some embodiments, the Spc105/KNL1 domain comprises at least 50% sequence similarity (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or any ranges there between) with the phosphodomain of Spc105 (SEQ ID NO:8) or KNL1 (SEQ ID NO:5) or a fragment thereof. In some embodiments, the Spc105/KNL1 domain comprises only conservative substitutions with respect to the phosphodomain of Spc105 (SEQ ID NO:8) or KNL1 (SEQ ID NO:5) or a fragment thereof.

In general, dimerization domains and elements (e.g., aka oligomerization domains and elements) that find use herein can be subdivided into two types: (1) domains/elements that are constitutive and (2) domains/elements that are regulated.

In some embodiments, constitutive domains/elements associate with their binding partner under suitable conditions (e.g., physiologically constitutive dimerization domains/elements will dimerize under physiologic conditions without the need for introduction of an initiator of dimerization). In some embodiments, a dimerization inducer is not required for dimerization of constitutive domains/elements. A skilled artisan will recognize that many heterologous domains whose associations are constitutive are well known in the art. Examples described in the art include, but are not limited to, heterodimerization of PDZ domains from the mammalian proteins neuronal nitric oxide synthase (nNOS) and syntrophin (Ung et al. (2001) EMBO J. 20: 3728-3737; herein incorporated by reference in its entirety), heterodimerization of the Xenopus XLIM1 and LDB1 proteins (Ung et al. (2001) EMBO J. 20: 3728-3737; herein incorporated by reference in its entirety), oligomerization of RFG (also named ELE1 or ARA70) through its coiled-coil domain (Monaco et al. (2001) Oncogene 20: 599-608; herein incorporated by reference in its entirety), oligomerization of the leucine zipper domain of yeast GCN4 (Harbury et al. (1993) Science 262: 1401-1407; herein incorporated by reference in its entirety), and oligomerization of the TEL helix-loop-helix (HLH) domain (Golub et al. (1996) Mol. Cell. Biol. 16, 4107-4116; herein incorporated by reference in its entirety) and their variants.

In some embodiments, regulated domains/elements associate with their binding partner in the presence of an inducer of dimerization. A skilled artisan will further recognize that many heterologous domains whose associations are regulated, rather than constitutive, are well known in the art. Examples include, but are not limited to, the Gyrase B-coumermycin system, the FKBP-rapamycin-FRAP system, and their variants. In some embodiments, these heterologous domains are domains from naturally occurring proteins or truncated active portions thereof. The binding domain can be small (e.g., <25 kDa), nonimmunogenic and accessible to cell permeable, nontoxic ligands.

Inducible/regulatable dimerization pairs include, for example, GyrB—GyrB (gyrase subunit B), FKBP—FRB (FK-binding protein—a domain (FRB) of the lipid kinase protein homologue FRAP (FKBP-rapamycin-associated protein)), FM—FM (F36M mutation of FK-binding protein), ToxT—ToxT (ToxT Protein of V. cholerae), DHFR—DHFR (dihydrofolate reductase), FKBP—FKBP (FK-binding protein), FKBP—Cyp (FK-binding protein—cyclophilin) and Cyp—Cyp (Cyclophilin). In one embodiment, bacterial Gyrase B polypeptide or fragments or variants thereof (e.g. amino acids 1-220 of E. coli GyrB) are induced to dimerize in the presence of coumermycin or a coumermycin analog (Farrar et al. (1996) Nature 383, 178-181 and Farrar et al., U.S. Pat. No. 6,916,846; herein incorporated by reference in their entireties). In another embodiment, a first functional domain is fused to the FRB (FRAP rapamycin binding) domain(s) or its variants of FRAP/mTOR or to an FKBP domain(s) or its variants of FKBP12 or its homologs, such that expression of the first functional domain fused to the FRB domain a dimer will form with a second functional domain fused to FKBP, or vice versa, in the presence of rapamycin or a rapamycin analog.

In some embodiments, both dimerization components are polypeptides or peptides (e.g., dimerization domains). Particular examples of such dimerizing polypeptides are GyrB, FM, ToxT, FKBP, and DI-FR. In other embodiments, one or both of the dimerization components is not a peptide or polypeptide.

In some embodiments, a first dimerization component is a polypeptide or peptide (e.g., dimerization domain) and a second dimerization component is a nucleic acid (e.g., dimerization element). Particular examples of such dimerizing pairs include, for example, E—ETR (MphR(A) protein and its operator ETR of E. coli), PIP—PIR (PIP protein of Streptomyces pristinaespiralis and its operator PIR), TetR—tetO (Tn10-derived tetracycline repressor TetR and its operator tetO), ArgR—argO (arginine-responsive repressor and its operator argO), ArsR—arsO (arsenic-responsive repressor and its operator arsO), HucR—hucO (uric acid-responsive repressor and its operator hucO), etc. Other such pairs are described by Ramos J. L. et al. (Microbiol MoI Biol Rev 69, 326-56, 2005) and Martinez-Bueno M. et al. (Bioinformatics 20, 2787-91, 2004); herein incorporated by reference in their entireties.

In some embodiments, a first dimerization component is a polypeptide or peptide (e.g., dimerization domain) and a second dimerization component is a small molecule (e.g., dimerization element). Particular examples of such dimerizing pairs include, for example, GyrB—coumarin antibiotics, FKBP—mTOR inhibitors, FRB—mTOR inhibitors, FM—mTOR inhibitors, Cyp—cyclosporins, Cyp-ascomycins, DIFR—antifolate, streptavidin—biotin analog, avidin—biotin analog, neutravidin—biotin analog, steroid hormone receptors—steroid hormones and analogs thereof, and ToxT—virstatin.

In other embodiments, neither component of a dimerization pair is a peptide or polypeptide.

Dimerization domains may be attached to functional domains via the formation of a fusion polypeptide comprising the two domains. Non-peptide/non-polypeptide dimerization elements may be attached to functional domains via direct covalent attachment or through a linker element.

Dimerization approaches and components are further described, for example, in Intl. Pat. App. WO 2009/146929; herein incorporated by reference in its entirety. Any other commercially-available dimerization components, or systems/methods for formation of dimers that are known in the filed may find use in some embodiments herein.

In some embodiments, the systems described herein comprise the capacity to tune the level of SAC activation. Various methods are available for such tunable activation, including: varying dimerization inducer concentration, presence or absence of phosphorylation inhibitor/enhancer, varying concentration of one or both constructs of a system, etc. In some embodiments, methods herein comprise administering a Spc105/KNL1 construct and a Mps1 construct to a system, cell, tissue, tumor, organism, etc., followed by tunably activating SAC toa desired level via administration of a desired (e.g., known or determined empirically) concentration of dimerization inducing agent.

In some embodiments, constructs are provided in which a functional domain (e.g., Spc105/KNL1 or Mps1 domain) is linked to a dimerization domain or element. In some embodiments, the functional domain and dimerization domain/element are directly connected. In other embodiments, a linker moiety connects the functional domain and dimerization domain/element. Suitable linkers may be peptide or polypeptide linkers (e.g., connecting a polypeptide functional domain to a peptide/polypeptide dimerization domain), or may be chemical linkers (e.g., connecting a polypeptide functional domain to a non-peptide/non-polypeptide dimerization element), such as a straight-chain or branched carbon chain, optionally comprising one or more functional groups (e.g., heteroatom-containing functional groups).

In some embodiments, provided herein are fusion polypeptides comprising a functional domain (e.g., Spc105/KNL1 or Mps1 domain) and a dimerization domain. In such embodiments, the two peptide/polypeptide domains may be directly connected (e.g., N-terminus to C-terminus) or may be connected via a peptide/polypeptide linker. An peptide/polypeptide linker may be of and suitable sequence and may confer one or more desirable characteristics to the fusion polypeptide, such as: solubility, spacing between domains, flexibility, etc. Peptide/polypeptide linkers are not limited to fusion polypeptide constructs; rather, they may also find use in other constructs within the scope herein, such as constructs comprising a functional domain (e.g., Spc105/KNL1 or Mps1 domain) and a non-peptide/non-polypeptide dimerization element.

In some embodiments, provided herein are functional domains (e.g., Spc105/KNL1 or Mps1 domain) connected to a peptide/polypeptide dimerization domain or a non-peptide/non-polypeptide dimerization element via a chemical linker moiety. In some embodiments, a chemical linker moiety comprises a straight or branched chain of 1-30 carbon atoms, optionally comprising one or more heteroatoms and branched or main-chain substituents. In some embodiments, the linker moiety comprises a multiatom straight or branched chain of atoms selected from C, H, N, O, P, and S. Functional groups comprising the linker moiety include, but are not limited to —CH₂—, ═CH—, =C═, CO, CONH, —NH₂, —OH, —SH, —O—, —S—, etc. In some embodiments, the linker moiety comprises one or more (CH₂)₂O groups or CONH groups.

In some embodiments, provided herein are systems comprising a first construct comprising a Mps1 domain and a first dimerization domain/element and a second construct comprising a Spc105/KNL1 domain and a second dimerization domain/element, wherein the first dimerization domain/element and the second dimerization domain/element are complementary such that they constitutively, or upon induction (e.g., by contact with an inducing agent), dimerize to form a complex (e.g., stable complex). In some embodiments, formation of the dimerization complex facilitates phosphorylation of the Spc105/KNL1 domain of the second construct by the Mps1 domain of the first construct. In some embodiments, in the absence of formation of the dimerization complex, little or no (e.g., below background) phosphorylation of the Spc105/KNL1 domain of the second construct occurs.

In some embodiments, formation of the dimerization complex results in a significant increase in phosphorylation of the Spc105/KNL1 domain of the second construct (e.g., 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 500-fold, 10³-fold, 10⁴-fold, 10⁴-fold, 10⁵-fold, 10⁶-fold, or more, or and suitable ranges there between). In some embodiments, the degree of increase in phosphorylation is proportional to the concentration of the first and/or second constructs. In some embodiments, the degree of activation of SAC is scalable/tunable based upon the concentration of the first and/or second constructs.

In some embodiments, induction of dimerization (e.g., by addition of a inducer of dimerization) results in a significant increase in phosphorylation of the Spc105/KNL1 domain of the second construct (e.g., 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 500-fold, 10³-fold, 10⁴-fold, 10⁴-fold, 10⁵-fold, 10⁶-fold, or more, or and suitable ranges there between). In some embodiments, the degree of increase in phosphorylation is proportional to the concentration of the first and/or second constructs. In some embodiments, the degree of activation of SAC is scalable/tunable based upon the concentration of the first and/or second constructs.

In some embodiments, methods are provided for activating the SAC in an in vitro or in vivo system (e.g., cell, tissue, tumor, organism, etc.) via the use of the Spc105/KNL1 and Mps1 dimerization constructs described herein. In some embodiments, tunable activation of the SAC is achieved by titrating (e.g., increasing/decreasing) the amount of Spc105/KNL1 dimerization construct, Mps1 dimerization construct, or dimerization inducer. In some embodiments, construct levels are controlled at the expression level. Systems and methods described herein are not limited by the routes of administering dimerization constructs and/or inducer or altering the concentrations thereof.

In many embodiments described herein, first and second constructs are provided for the activation (e.g., inducible activation, tunable activation, etc.) of SAC. In some embodiments, provided herein is a single construct comprising: (i) a Mps1 domain, (ii) a Spc105/KNL1 domain, and (iii) one or more domains, elements, or moieties configured to facilitate phosphorylation (e.g., tunable phosphorylation) of the Spc105/KNL1 domain by the Mps1 domain. For example, in some embodiments, Mps1 and Spc105/KNL1 domains are located at disparate locations in the primary sequence of a polypeptide fusion, such that phosphorylation of the Spc105/KNL1 domain by the Mps1 domain does not occur or occurs at a low rate. However, upon induction of dimerization or a pair of dimerization domains/elements located on the fusion polypepeitde, interaction of the Mps1 and Spc105/KNL1 domains is induced, phosphorylation of the Spc105/KNL1 domain by the Mps1 domain occurs, and SAC is activated. Any suitable orientation of Mps1 domain, Spc105/KNL1 domain, and dimerization domains/elements is within the scope herein (e.g., (Mps1 domain)-(dimerization domain 1)-(linker)-(dimerization domain 2)-(Spc105/KNL1 domain); (dimerization domain 1)-(Mps1 domain)-(linker)-(Spc105/KNL1 domain)-(dimerization domain 2); etc.).

Embodiments herein contemplate the delivery of exogenous nucleic acids encoding SAC-activating dimerization constructs, or the delivery of proteins themselves (e.g., recombinant SAC-activating dimerization constructs, etc.) to a system, cell, tissue, tumor, subject, etc. via any suitable method.

In some embodiments, nucleic acids are delivered within suitable vectors. The present invention is not limited to any particular vector. Indeed, a variety of vectors may be used to deliver the nucleic acids.

In certain embodiments, the nucleic acids are delivered via an adenovirus vector. (See e.g., Westfall et al., Meth. Cell Biol. 32:307-322 (1998); and U.S. Pat. Nos. 6,451,596, 6,083,750, 6,063,622, 6,057,158, or 5,994,132, all of which are herein incorporated by reference). In some embodiments, a nucleic acid encoding a construct(s) described herein are delivered via an adeno-associated vector (AAV). In some embodiments, the AAV vector integrates into the genome of the cells to which it is administered (e.g., a patient's cells (e.g., endothelial cells)). A number of AAV vectors which have been developed for gene therapy are useful in the present invention (See e.g., U.S. Pat. Nos. 5,173,414; 5,139,941; and 5,843,742; PCT publications WO92/01070 and WO93/03769; Lebkowski et al., Mol. Cell. Biol. 8:3988-3996 (1988); Carter, Curr. Opin. Biotech. 3:533-39, (1992); Muzyczka, Curr. Top, Microbiol. Immunol. 158:97-129, (1994); Kotin, Human Gene Ther. 5:793-801, (1994); Shelling and Smith, Gene Ther. 1:165-69, (1994); Zhou et al., J. Exp. Med. 179:1867-1875, (1994); U.S. Pat. Nos. 6,451,596, 6,083,750, 6,063,622, 6,057,158, or 5,994,132; Ferrari et al., Nature Med. 3(11):1295-97, (1997); and Gregorevic et al., Nature. Med. 10(8): 828 (2004), each of which is incorporated herein by reference in its entirety).

In some embodiments, recombinant adenovirus vectors are constructed by homologous recombination of a shuttle vector containing a nucleic acid encoding one or more SAC-activating dimerization constructs and the full-length adenovirus DNA following co-transfection into a cell line. In some embodiments, the full-length adenovirus DNA is provided from pJM17 which is a 0-100 map unit (m.u.) derivative of adenovirus serotype (Ad5) that contains a partial deletion in the E3 region and a 4.3-kb pBRX insert at 3.7 m.u. (See e.g., Graham and Prevec, Manipulation of Adenovirus Vectors, in Gene Transfer and Expression Protocols, E. J. Murray ed., Humana, Clifton, N.J. (1991); and Becker et al., Use of Recombinant Adenovirus for Metabolic Engineering of Mammalian Cells, in Methods in Cell Biology, Vol 43 M. G. Roth ed., Academic Press, N.Y. (1994); Grahm and Prevec, Methods Mol. Biol. 7, 109 (1991); herein incorporated by reference in their entireties). In some embodiments, a shuttle vector comprises 0-1 m.u. and 9-16 m.u. of the Ad5 genome flanking an expression cassette containing the nucleic acid encoding one or more SAC-activating dimerization constructs. Embodiments herein are not limited by the type of AAV or the methods of construction thereof.

In other embodiments, the nucleic acid encoding SAC-activating dimerization constructs are delivered via a liposome or naked DNA plasmid. In some embodiments, the liposome is a cationic liposome (See e.g., U.S. Pat. Nos. 5,908,777 and 5,676,954 each incorporated herein by reference in their entireties; Hug and Sleight, Biochim. Biophys. Acta. 1097:1-17, (1991); Straubinger et al., in Methods of Enzymology, Vol. 101 pp. 512-527 (1993); Felgner et al., Nature 337:387-388, (1989); and Felgner et al., PNAS (1987) 84:7413-7416) (1987); herein incorporated by reference in their entireties). An example of a commercially available cationic liposome carrier useful in the present invention is LIPOFECTIN (Bethesda Research Laboratories Life Technologies, Inc., Gaithersburg Md.).

In some embodiments, vector comprising nucleic acid encoding one or more SAC-activating dimerization constructs further includes a suitable promoter (e.g., cell specific promoter, etc.) and/or enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5′ flanking nontranscribed sequences. In other embodiments, DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required non-transcribed genetic elements.

In some embodiments, nucleic acid constructs comprise elements for introduction of the Mps1 and Spc105/KNL1 constructs described herein via a CRISPR/Cas system (See, e.g., WO 2014093661, WO 2013176772, etc.; herein incorporated by reference in their entireties).

In some embodiments, the DNA sequence in an expression vector is operatively linked to an appropriate expression control sequence(s) (e.g., promoter) to direct mRNA synthesis. In some embodiments, the promoter is the cytomegalovirus (CMV) promoter. Other promoters useful in embodiments of the present invention include, but are not limited to, the LTR or SV40 promoter, the E. coli. lac or trp, the phage lambda PL and PR, T3 and T7 promoters, HSV thymidine kinase, and mouse metallothionein-I promoters and other promoters known to control expression of genes in prokaryotic or eukaryotic cells (e.g., endothelial cells) or their viruses. In some embodiments, recombinant expression vectors include selectable markers permitting transformation of the host cell (e.g. dihydrofolate reductase or neomycin resistance for eukaryotic cell culture). In some embodiments, the promoter is a tissue specific and/or inducible promoter. In some embodiments, the promoter is regulated by an exogenous factor (e.g., diet, light, activator agent, etc.).

In some embodiments, transcription of the DNA encoding peptides and/or polypeptides described herein by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription; Enhancers useful in the present invention include, but are not limited to, the SV40 enhancer (e.g., 100 to 270 base pairs on the late side of the replication origin), a cytomegalovirus early promoter enhancer, the polyoma enhancer (e.g., on the late side of the replication origin), and adenovirus enhancers.

In other embodiments, the expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. In still other embodiments of the present invention, the vector includes appropriate sequences for amplifying expression.

In some embodiments, systems and methods are provided herein for the treatment of cancers or other diseases of conditions caused by of linked to aneuploidy or other chromosome separation abnormalities. In some embodiments, the Mps1 and Spc105/KNL1 polypeptide constructs described herein (or nucleic acids encoding such constructs) are used as therapeutics for the treatment or prevention of cancer, pre-cancer, metastasis, etc.

In some embodiments, systems and methods are provided for reducing or inhibiting the proliferation of cancer (e.g., tumor) cells in an individual. In some embodiments, an individual is identified on the basis that the individual is known to have, or be at risk of (e.g., based on prior occurance), an aneuploid cancer (e.g., tumor) and administering to said individual an effective amount of the compositions and/or systems herein to reduce or inhibit the proliferation of the cancer (e.g., tumor) cells in the aneuploid cancer (e.g., tumor). In some embodiments, compositions and/or systems herein are administered to an individual who has cancer. In some embodiments, the individual may not have been identified as having an aneuploid cancer (e.g., the cancer was not evaluated for aneuploidy or the cancer did not show present signs of aneuploidy).

In some embodiments, methods for determining if an individual should be administered an systems and/or compositions described herein are provided. In some embodiments, a cancer (e.g., tumor) sample is obtained from an individual and a karyotype analysis on the sample is performed to determine if the cancer (e.g., tumor) contains cells that are aneuploid. The presence of aneuploid cells in the cancer (e.g., tumor) sample indicates the cells should be treated as described herein. In some embodiments, Mps1 and/or Spc105/KNL1 constructs administered to the individual. The sample may be obtained from the individual by performing a biopsy. In some embodiments, the sample may be a DNA sample or a cellular sample. According to some aspects of the invention, methods of reducing or inhibiting cancer (e.g., tumor) cells lacking a functional endogeneous tumor suppressor gene (e.g., with a mutation or deletion of one or both alleles) are provided. In some embodiments, cancer (e.g., tumor) cells lacking a functional endogeneous tumor suppressor gene are contacted with an effective amount therapeutic compositions or systems herein. In some embodiments, the tumor suppressor gene is p53. In some embodiments, the individual is known to have one or more mutations in one or more oncogenes, such as ras, c-myc, erB-2, src, and bcl-2. In some embodiments, the individual is at risk of developing cancer or has been previously diagnosed with cancer. In some embodiments, the individual has one or more other indicia or risk factors for a disease or condition associated with aneuploidy.

Without being bound by theory, it should be appreciated that diseased tissues (e.g., tumor or cancer tissue) associated with aneuploid cells may entirely comprise aneuploid cells, may contain a subset of aneuploid cells (e.g., about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or higher or lower percentages of aneuploid cells) as aspects of the invention are not limited in this respect. In some embodiments, the aneuploid cells may be homogeneous (all have the same genetic defects, for example the same genomic deletion, duplications, or combinations thereof). In some embodiments, the aneuploid cells may be heterogeneous (e.g., different cells or subsets of cells have different genetic defects, for example different extents of chromosomal deletions, duplications, or combinations thereof). It should be appreciated that certain diseases or conditions are associated with genomic instability leading to increasing levels of aneuploidy (e.g., larger amounts of genetic abnormalities within each aneuploid cells and/or more cells that are aneuploid) over time. Accordingly, aspects herein may be useful to treat subjects that have risk factors (e.g., one or more cancer-associated mutations) and/or indicia (e.g., low levels of genetic deletions and/or duplications) prior to the development of significant levels of aneuploidy (e.g., to prevent, reduce, or delay the development, growth or proliferation of aneuploid cells).

In some embodiments, aneuploidy is detected through karyotyping. Other techniques include Fluorescence In Situ Hybridization (FISH), Quantitative Polymerase Chain Reaction (PCR) of Short Tandem Repeats, Quantitative Fluorescence PCR (QF-PCR), Quantitative Real-time PCR (RT-PCR) dosage analysis, Quantitative Mass Spectrometry of Single Nucleotide Polymorphisms, Spectral karyotype analysis (SKY), and Comparative Genomic Hybridization (CGH). In some embodiments, karyotype analysis is performed on a cancer (e.g., tumor) sample that has been obtained from an individual. Tumor tissue removed from an individual by a biopsy can be used as a tumor sample. In some embodiments, the cancer (e.g., tumor) sample is a cellular sample or a DNA sample. Embodiments herein are not limited to the methods of detecting aneuploidy and that any method which allows the determination of aneuploidy can be used. As used herein, an individual includes a mammal, such as a human, non-human primate, cow, rabbit, horse, pig, sheep, goat, dog, cat, or rodent such a rat, mouse or a rabbit. In some embodiments, the individual is a human. In some embodiments, the methods are employed to reduce or inhibit the proliferation of the tumor or the unwanted mammalian cell proliferation in an individual, such as a mammal (e.g., human).

Systems, compositions, and methods of the invention are useful for treating diseased conditions in which subset of cells in an individual are aneuploid, such as certain tumors, cancers, neurological disorders such as Alzheimer's disease, and/or unwanted mammalian proliferation of aneuploid cells. Tumors treatable by the compounds of the invention include, for example, benign and malignant solid tumors, and benign and malignant non-solid tumors. Examples of solid tumors include but are not limited to: biliary tract cancer, brain cancer (including glioblastomas and medulloblastomas), breastcancer, cervical cancer, choriocarcinoma, colon cancer, endometrial cancer, esophageal cancer, gastric cancer, intraepithelial neoplasms, including Bowen's disease and Paget's disease, liver cancer, lung cancer, lymphomas, including Hodgkin's disease and lymphocytic lymphomas, neuroblastomas, oral cancer, including squamous cell carcinoma, ovarian cancer, including those arising from epithelial cells, stromal cells, germ cells and mesenchymal cells, pancreatic cancer, prostate cancer, rectal cancer, renal cancer including adenocarcinoma and Wilms tumor, sarcomas, including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma and osteosarcoma, skin cancer, including melanoma, Kaposi's sarcoma, basocellular cancer and squamous cell cancer, testicular cancer, including germinal tumors (seminomas, and non-seminomas such as teratomas and choriocarcinomas), stromal tumors and germ cell tumors, and thyroid cancer, including thyroid adenocarcinoma and medullary carcinoma. In some embodiments, the tumor is non-pancreatic. Examples of non-solid tumors include but are not limited to hematological neoplasms. A hematologic neoplasm includes, for example, lymphoid disorders, myeloid disorders, and AIDS associated leukemias. Lymphoid disorders include but are not limited to acute lymphocytic leukemia and chronic lymphoproliferative disorders (e.g., lymphomas, myelomas, and chronic lymphoid leukemias). Lymphomas include Hodgkin's disease and non-Hodgkin's lymphoma. Chronic lymphoid leukemias include T cell chronic lymphoid leukemias and B cell chronic lymphoid leukemias. Myeloid disorders include chronic myeloid disorders such as for instance, chronic myeloproliferative disorders, myelodysplastic syndrome and acute myeloid leukemia. Chronic myeloproliferative disorders include but are not limited to angiogenic myeloid metaplasia, essential thrombocythemia, chronic myelogenous leukemia, polycythemia vera, and atypical myeloproliferative disorders. Atypical myeloproliferative disorders include, for example, atypical Chronic Myelogenous Leukemia (CML), chronic neutrophilic leukemia, mast cell disease, and chronic eosinophilic leukemia. Conditions of unwanted mammalian cell proliferation and treatable by this invention include familial adenomatous polyposis, dysplasia, hyperplasia (e.g., benign prostatic hyperplasia), fibrotic disorders, arteriosclerotic disorders, and dermatological disorders.

In some embodiments, provided herein are methods of reducing or inhibiting the proliferation of cancer (e.g., tumor) cells lacking one or more functional tumor suppressor gene(s). Tumor suppressor genes are genes which, in their wild type alleles, express proteins that suppress abnormal cell proliferation. Mutations of tumor suppressor genes can lead to loss of functional tumor suppressor protein expression and consequently, abnormal cell proliferation which may be accompanied by aneuploidy. In some embodiments, loss of tumor suppressor activity leads to aneuploidy. Examples of tumor suppressor genes include, but are not limited to, the retinoblastoma susceptibility gene or RB gene, the protein 53 (p53) gene (NM_000546.4; GI: 187830767; also known as antigen NY-CO-13, phosphoprotein p53, transformation-related protein 53 (TRP53), tumor suppressor p53), the deleted in colon carcinoma (DCC) gene (NM 005215.3; GI:260436868; also known as colorectal cancer suppressor) and the neurofibromatosis type 1 (NF-1) tumor suppressor gene (NM OO 1042492.2; GI:270132520). In some embodiments, methods comprise: identifying an individual with a tumor suppressor defect known to be associated with cancer and administering to the individual an effective amount of the therapeutic systems and compositions described herein. Methods to determine the suppressor or oncogene status of a tumor are known in the art and may involve mutational analysis by sequencing, DNA analysis, RNA analysis, and protein analysis

The Spc105/KNL1 and/or Mps1 polypeptide dimerization constructs (or nucleic acids encoding such constructs) described herein may be administered to a subject per se or in the form of a pharmaceutical composition. Pharmaceutical compositions may be manufactured by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions may be formulated in conventional manner using one or more physiological acceptable carriers, diluents, excipients, or auxiliaries which facilitate processing of the therapeutic compositions into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

For topical administration the compounds of the invention may be formulated as solutions, gels, ointments, creams, suspensions etc. as are well-known in the art.

Systemic formulations include those designed for administration by injection, e.g. subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal, oral or pulmonary administration.

For injection, therapeutic compositions may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. The solution may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Alternatively, therapeutic compositions may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For oral administration, therapeutic compositions may be readily formulated by combining with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquid gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. For oral solid formulations such as, for example, powders, capsules and tablets, suitable excipients include fillers such as sugars, such as lactose, sucrose, mannitol and sorbitol; cellulose preparations such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium, carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP); granulating agents; and binding agents. If desired, disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidine, atgar, or alginic acid or a salt thereof such as sodium alginate. If desired, solid dosage forms may be sugar-coated or enteric-coated using standard techniques. For oral preparations such as, for example, suspensions, elixirs and solutions, suitable carriers, excipients or diluents include water, glycols, oils, alcohols, etc. Additionally, flavoring agents, preservatives, coloring agents and the like may be added.

For buccal administration, the compounds may take the form of tablets, lozenges, etc. formulated in conventional manner.

For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount.

Therapeutic compositions may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

Alternatively, other pharmaceutical delivery system may be employed. Liposomes and emulsions are well known examples of delivery vehicles. Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity. Additionally, therapeutic compositions may be delivered using a sustained-release system, such as semipermeable matrices of solid polymers containing the therapeutic agent. Various of sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.

The Spc105/KNL1 and/or Mps1 polypeptide dimerization constructs (or nucleic acids encoding such constructs) described herein will generally be used in an amount effective to achieve the intended purpose. For use to treat or prevent cancer, therapeutic compositions are administered or applied in a therapeutically effective amount. By therapeutically effective amount is meant an amount which is effective to ameliorate, or prevent the symptoms of the disease or disorder, or prolong the survival of the patient being treated. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

For systemic administration, a therapeutically effective dose can be estimated initially from in vitro assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell culture (i.e. the concentration of test compound that inhibits 50% of Survivin dimerization). Such information can be used to more accurately determine useful doses in humans.

In some embodiments, one or more chemotherapeutics or other cancer therapies are provided as co-therapies with Spc105/KNL1 and/or Mps1 polypeptide dimerization constructs (or nucleic acids encoding such constructs) described herein, with or without (known) synergism between the co-administered therapies.

In some embodiments, exemplary anticancer agents suitable for co-administeration include, but are not limited to: 1) alkaloids, including microtubule inhibitors (e.g., vincristine, vinblastine, and vindesine, etc.), microtubule stabilizers (e.g., paclitaxel (Taxol), and docetaxel, etc.), and chromatin function inhibitors, including topoisomerase inhibitors, such as epipodophyllotoxins (e.g., etoposide (VP-16), and teniposide (VM-26), etc.), and agents that target topoisomerase I (e.g., camptothecin and isirinotecan (CPT-11), etc.); 2) covalent DNA-binding agents (alkylating agents), including nitrogen mustards (e.g., mechlorethamine, chlorambucil, cyclophosphamide, ifosphamide, and busulfan (MYLERAN), etc.), nitrosoureas (e.g., carmustine, lomustine, and semustine, etc.), and other alkylating agents (e.g., dacarbazine, hydroxymethylmelamine, thiotepa, and mitomycin, etc.); 3) noncovalent DNA-binding agents (antitumor antibiotics), including nucleic acid inhibitors (e.g., dactinomycin (actinomycin D), etc.), anthracyclines (e.g., daunorubicin (daunomycin, and cerubidine), doxorubicin (adriamycin), and idarubicin (idamycin), etc.), anthracenediones (e.g., anthracycline analogues, such as mitoxantrone, etc.), bleomycins (BLENOXANE), etc., and plicamycin (mithramycin), etc.; 4) antimetabolites, including antifolates (e.g., methotrexate, FOLEX, and MEXATE, etc.), purine antimetabolites (e.g., 6-mercaptopurine (6-MP, PURINETHOL), 6-thioguanine (6-TG), azathioprine, acyclovir, ganciclovir, chlorodeoxyadenosine, 2-chlorodeoxyadenosine (CdA), and 2′-deoxycoformycin (pentostatin), etc.), pyrimidine antagonists (e.g., fluoropyrimidines (e.g., 5-fluorouracil (ADRUCIL), 5-fluorodeoxyuridine (FdUrd) (floxuridine)) etc.), and cytosine arabinosides (e.g., CYTOSAR (ara-C) and fludarabine, etc.); 5) enzymes, including L-asparaginase, and hydroxyurea, etc.; 6) hormones, including glucocorticoids, antiestrogens (e.g., tamoxifen, etc.), nonsteroidal antiandrogens (e.g., flutamide, etc.), and aromatase inhibitors (e.g., anastrozole (ARIMIDEX), etc.); 7) platinum compounds (e.g., cisplatin and carboplatin, etc.); 8) monoclonal antibodies (e.g., conjugated with anticancer drugs, toxins, and/or radionuclides, etc.; neutralizing antibodies; etc.); 9) biological response modifiers (e.g., interferons (e.g., IFN-.alpha., etc.) and interleukins (e.g., IL-2, etc.), etc.); 10) adoptive immunotherapy; 11) hematopoietic growth factors; 12) agents that induce tumor cell differentiation (e.g., all-trans-retinoic acid, etc.); 13) gene therapy techniques; 14) antisense therapy techniques; 15) tumor vaccines; 16) therapies directed against tumor metastases (e.g., batimastat, etc.); 17) angiogenesis inhibitors; 18) proteosome inhibitors (e.g., VELCADE); 19) inhibitors of acetylation and/or methylation (e.g., HDAC inhibitors); 20) modulators of NF kappa B; 21) inhibitors of cell cycle regulation (e.g., CDK inhibitors); and 22) modulators of p53 protein function.

In some embodiments, the co-administered agents are formulated into a single dose and/or composition. In some embodiments, the co-administered agents are in separate doses and/or compositions. In some embodiments in which separate doses and/or compositions are administered, the doses and/or compositions are administered simultaneously, consecutively, or spaced over a time span (e.g., <30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, or more, or any suitable ranges therebetween).

In some embodiments, systems, compositions and methods herein find use in the prevention of aneuploidy in cells in vitro. In some embodiments, the Mps1 and Spc105/KNL1 constructs (and optionally dimerization inducer) are administered or introduced into cultured cells (e.g., for research or clinical uses). In some embodiments, cells comprise induced pluripotent stem cells (iPSs) or embryonic stem cells (ESCs). In some embodiments, methods herein reduce the occurrence of aneuploidy in ECSs and iPSs in culture. In some embodiments, following treatment or introduction of the Mps1 and Spc105/KNL1 constructs and systems herein into ECSs and/or iPSs, the cells are administered or introduced (e.g., therapeutically) into a subject for treatment of a disease of condition. In some embodiments, the methods and systems described herein reduce aneuploidy and cancers resulting the transplantation of iPSs and/or ECSs into a subject.

EXPERIMENTAL Example 1 Mps1, Artificially Localized to the Kinetochore, Phosphorylates Spc105 and Activates the SAC

Experiments were conducted during development of embodiments of the present invention to determine whether microtubule attachment to the kinetochore silences the SAC by promoting the dissociation of SAC proteins from the kinetochore (FIG. 1 a ), and whether engineering persistent localization of key SAC proteins at the kinetochore constitutively activates the SAC. Rapamycin-induced dimerization of 2xFkbp12 and Frb was used to artificially localize or ‘anchor’ key phosphoregulators and SAC proteins: Mps1, Ipl1 (Aurora B), Glc7 (PP1) or Mad1 within the kinetochore (FIG. 1 b ). In the absence of rapamycin, each Frb-tagged protein retained its normal cellular distribution. Addition of rapamycin to the culture media rapidly anchored it to the kinetochore subunit tagged with 2xFkbp12 (FIG. 1 c and FIG. 2 a ).

Mps1 anchored at Mtw1-C in this manner led to the accumulation of large-budded cells that were arrested in metaphase (FIG. 1 d, e ). The kinetochores in these cells recruited both Bub1 and Mad1, indicating that the arrest was mediated by the SAC (FIG. 1 d ). These observations are consistent with findings that Mps1 fused to kinetochore proteins activates the SAC (refs 10, 11; herein incorporated by reference in their entireties). Other SAC proteins tested: Ipl1, Mad1 and Glc7, did not delay the cell cycle when anchored to Mtw1-C(FIG. 2 b ). The phosphorylation of the kinetochore protein Spc105 at one or more of its conserved ‘MELT’ motifs was necessary for the anchored Mps1 to activate the SAC (ref. 12; herein incorporated by reference in its entirety; FIG. 1 e ). These effects did not require the kinase activity of Ipl1, indicating that the anchored Mps1 did not activate the SAC indirectly by disrupting either microtubule attachment or force generation (ref. 13; herein incorporated by reference in its entirety; FIG. 2 c, d ). This is consistent with data from other organisms and with the dispensability of Ipl1 for SAC signalling in budding yeast (refs. 14, 15; herein incorporated by reference in their entireties). Thus, anchoring Mps1 to the kinetochore is sufficient for inducing constitutive SAC signaling.

SAC Proteins that Act Downstream from Mps1 can Function within Attached Kinetochores

The above experiments were performed in asynchronous yeast cultures. Consequently, it could not ascertained whether the anchored Mps1 activated the SAC mostly in prometaphase, before all kinetochores attach to microtubules, or if Mps1 can reactivate the SAC when anchored within stably attached kinetochores. To test this, CDC20, the gene that encodes the activating subunit of the anaphase-promoting complex (APC), was repressed to prevent yeast cells from entering anaphase even after all of the kinetochores were attached and the SAC was satisfied. Mps1 was anchored at Mtw1-C in such cells, released them from the arrest by inducing CDC20 expression, and then monitored cell-cycle progression (FIG. 3 a ). It was found that cells that had Mps1 anchored at Mtw1-C underwent a persistent cell-cycle arrest, whereas control cells completed anaphase within 20 min (FIG. 3 a ). Thus, Mps1 reactivates the SAC, when it is anchored to kinetochores with stable microtubule attachments.

These results demonstrate that SAC proteins downstream from Mps1 bind to and function from attached kinetochores. No significant changes were detected when the nanoscale separation between key kinetochore domains in metaphase and rapamycin-treated cells were compared using high-resolution co-localization (FIG. 3 b ). This indicates that if architectural changes that facilitate SAC protein binding do occur, they do so when the kinetochore is attached. these data demonstrates that microtubule attachment to the kinetochore hampers either Mps1 localization to the kinetochore or its kinase activity to silence the SAC.

Endogenous Mps1 Binds to Attached Kinetochores

Mps1 gradually disappears from the kinetochore clusters as yeast cells progress from prometaphase to metaphase (FIG. 2 e ). However, Mps1 is targeted for degradation by the APC (ref 17; herein incorporated by reference in its entirety). Upon inactivation of the APC using CDC20 repression, Mps1-Frb-GFP autonomously localized to attached kinetochores (FIG. 3 c , left). The autonomously localized Mps1 did not activate the SAC, because both Bub3 and Mad1 were absent from the kinetochores (FIG. 3 c , right). Furthermore, these cells entered anaphase without any detectable delay following release from the metaphase block (FIG. 3 a , dotted line). Mps1 was present at the kinetochore even as these cells entered anaphase (FIG. 2 f ). Thus, the removal of Mps1 from the kinetochore is not necessary for either SAC silencing or anaphase onset.

Mps1 molecules that autonomously localize to attached kinetochores do not activate the SAC, but a similar number of Mps1 molecules anchored at Mtw1-C activate it constitutively (FIG. 2 e ). It is contemplated that the inability of the autonomously localized Mps1 to activate the SAC is due to: inability to reach and phosphorylate Spc105 from its endogenous binding position in the kinetochore; the inhibition of the kinetochore-bound Mps1 kinase; or the upregulation of Glc7 phosphatase activity in attached kinetochores (refs. 18, 19; herein incorporated by reference in their entireties). Upregulation of Glc7 activity is unlikely to be the main mechanism that silences the SAC, because Glc7 is not necessary for anaphase onset19. Therefore, experiments were conducted during development of embodiments herein to investigate how microtubule attachment affects Mps1 kinase activity within the kinetochore.

The Ability of Mps1 to Activate the SAC Depends on its Position within the Kinetochore

It was tested whether the binding position of Mps1 within the kinetochore can affect its ability to phosphorylate Spc105 and initiate SAC signaling. In metaphase, the budding yeast kinetochore spans ˜80 nm along its longitudinal axis, from the amino terminus of Ndc80 to the centromeric nucleosome (ref. 20; herein incorporated by reference in its entirety). It contains ˜8 copies of the Ndc80 complex and Spc105 molecules distributed with an average inter-molecular spacing of ˜8 nm around the microtubule circumference (refs. 21, 22; herein incorporated by reference in their entireties), and with little inter-molecular staggering along the length of the microtubule (ref 23; herein incorporated by reference in its entirety) (FIG. 1 b ). This architecture indicates that the proximity of Mps1 to Spc105 along the longitudinal axis of the kinetochore affects its ability to phosphorylate Spc105.

To reveal the position-specific activity of Mps1, rapamycin-induced dimerization stably anchors and confines it at specific kinetochore positions. This was determined using three measurements (FIG. 4 a-c ). First, it was found that the anchoring was stable, as indicated by negligible turn-over of Mps1-Frb-GFP anchored at Ndc80-C (FIG. 4 a ). Although this high stability is ideal for studying positions specific activity, it may be non-physiological 24. Second, Forster resonance energy transfer (FRET) measurements indicated that the anchored protein is confined within a 10 nm region around the anchoring point (FIG. 4 b ). Finally, the total number of molecules anchored within the kinetochore was determined by the abundance of the anchored protein (ref. 25; herein incorporated by reference in its entirety) and also its kinetochore anchor (ref 21; herein incorporated by reference in its entirety) (FIG. 4 c ). As a result, the entire nuclear pool of low-abundance proteins such as Mps1 and Ipl1 was anchored at the selected kinetochore position.

Mps1 was constitutively anchored at six distinct positions selected to sample the 80 nm length of the kinetochore-microtubule attachment (FIG. 4 d , top). To assess the effects of anchoring Mps1 on the cell cycle, an equal number of cells were plated on control plates and on plates containing rapamycin, and compared the number of colonies formed in each case (FIG. 4 d , right). These experiments were performed in heterozygous diploids that also expressed wild-type Mps1, because Mps1 activity is also essential for other cellular functions (ref 26; herein incorporated by reference in its entirety). Even though the wild-type, diffusible Mps1 provides these essential functions, it is not required for SAC activation (FIG. 5 a, b ). Furthermore, haploids expressing only Mps1-Frb also exhibited identical SAC activation phenotypes (FIG. 5 c ).

When Mps1 was constitutively anchored at four different locations within the inner kinetochore, ranging from Ndc80-C to Ctf19-C, it completely inhibited colony growth (FIG. 4 d ). MAD2 deletion restored colony growth, indicating that the lack of growth was due to constitutive SAC activation (FIGS. 4 d and 6 a ). Mps1 anchored at two positions located in the outer kinetochore, N-Ndc80 and Ask1-C(a Dam1 complex subunit), had no effect on colony growth (FIG. 4 d ). Although the number of Mps1 molecules anchored in the inner kinetochore positions was 30-50% higher than the number of Mps1 molecules anchored in the outer kinetochore, these differences did not strictly correlate with SAC activation phenotypes (FIG. 6 b ). Reducing the length of the linker between Mps1 and Frb-GFP did not affect the observed phenotypes (FIG. 6 c ). Finally, the observed effects were specific to Mps1: constitutive anchoring of Ipl1 or Mad1 at the same positions did not result in the same phenotypes (FIG. 6 d-g ).

These data demonstrate that the position of Mps1 within the kinetochore affects its ability to activate the SAC. As Mps1 phosphorylates Spc105 to activate the SAC, the observed phenotypes likely reflect whether or not the anchored Mps1 can access the phosphodomain of Spc105. It is also notable that Mps1 activates the SAC from different locations over a 30 nm span20 (the metaphase separation between Ndc80-C and Ctf19-C), even though its kinase activity is spatially confined to individual anchoring locations. It is contemplated that to encounter the confined kinase activity over this wide span, the long and unstructured phosphodomain of Spc105 assumes variable conformations.

Mps1 Anchored in the Outer Kinetochore does not Activate the SAC

To confirm that the inability of Mps1 to activate the SAC from the outer kinetochore is due to inability to phosphorylate Spc105, the effects were characterized of anchoring Mps1 to the carboxy termini of seven other subunits of the heterodecameric Dam1 complex (ref. 27; herein incorporated by reference in its entirety) (FIG. 7 a ). In addition to Ask1, Mps1 anchored to three other Dam1 subunits did not affect the colony growth (FIGS. 7 b and 8 a ). Mps1 anchored to four other subunits delayed colony formation, but did not seem to affect the number of colonies formed (FIGS. 7 b and 8 a ). It is contemplated that low colony growth was due to a transient SAC-mediated delay in the cell cycle (FIGS. 7 c and 5 c ). As before, reduced length of the flexible linker fusing the Mps1 kinase domain to Frb did not affect the observed cell-cycle delay (FIG. 8 b ).

Experiments were conducted during development of embodiments herein to determine whether the anchored Mps1 perturbed Dam1 complex localization and function, because Dam1 subunits are known Mps1 substrates (refs. 27, 28; herein incorporated by reference in their entireties). Distribution of Dad4 was quantified over the mitotic spindle after anchoring Mps1 to other Dam1 subunits (FIG. 7 d ). Dad4-mCherry co-localized with the anchored Mps1-Frb-GFP in every case, and its distribution was indistinguishable from Dad4 distribution in untreated cells. Thus, the association of the Dam1 complex with the kinetochore remained unaffected. The separation between kinetochore clusters in rapamycin-treated cells was also indistinguishable from the corresponding length in untreated cells (FIG. 7 e ). This indicates that force generation at the kinetochore, a process in which the Dam1 complex is the dominant contributor, was not affected (ref. 29; herein incorporated by reference in its entirety). Thus, the anchored Mps1 does not perturb Dam1 complex function, and the observed phenotypes reflect whether or not the anchored Mps1 can phosphorylate Spc105.

This is because dimensions of the Dam1 complex (ref. 27; herein incorporated by reference in its entirety) and its narrow distribution along the length of the kinetochore-microtubule attachment (ref. 23; herein incorporated by reference in its entirety) indicate that all of the anchoring points are confined within a ˜10-nm-wide zone. Although the structure of the Dam1 complex is unknown, data are consistent with the C termini of Dam1 subunits facing towards or away from the centromere (FIG. 7 f , arrows). It is contemplated that this orientation constrains the orientation of the anchored Mps1, and determines whether or not Mps1 phosphorylates Spc105 to activate the SAC.

Phosphorylation of Spc105 by Mps1 is Sufficient to Initiate SAC Signaling

The physical proximity between the Mps1 kinase and the phosphodomain of Spc105 controls the state of the SAC. Therefore, experiments were conducted to determine whether a forced interaction between the two outside the kinetochore is sufficient to activate the SAC. We engineered a minimal, anchorable phosphodomain comprising residues 120-329 of Spc105 (referred to as Spc105120: 329, FIG. 9 a ). It contains all 6 MELT motifs, but no known kinetochore-binding activity. When Spc105120: 329 was anchored to Mps1-Fkbp12 in asynchronously dividing cells, the cells arrested in metaphase (FIG. 9 b ). Spc105120: 329 also localized to kinetochore clusters under these conditions and recruited Mad1 (FIGS. 9 c and 10 a ). The kinetochore localization of Mad1 and Spc105120: 329, when the latter anchored to Mps1, is mediated by Mps1 binding to the kinetochores. MAD2 deletion abolished the cell-cycle arrest indicating that the arrest resulted from SAC activation (FIG. 9 b , dashed line). When Spc105120: 329:6A, the non-phosphorylatable version of Spc105120: 329, was anchored to Mps1, it did not activate the SAC (FIG. 9 b, c ). Thus, the phosphorylation of MELT motifs in Spc105120: 329 by Mps1 is necessary for the observed cell-cycle arrest.

To examine whether kinetochores contributed to the SAC signaling, cells carrying ndc10-1, a temperature-sensitive allele of the gene encoding the centromeric protein Ndc10 (ref. 30; herein incorporated by reference in its entirety), were used. At the restrictive temperature, these cells do not assemble functional kinetochores, and are thus unable to activate the SAC. However, when Spc105120: 329 was anchored to Mps1 at the restrictive temperature, ndc10-1 cells experienced a cell-cycle delay similar to the delay seen in NDC10 cells under the same conditions (FIG. 10 b ). Thus, the SAC signaling induced by the forced interaction between Spc105120: 329 and Mps1 does not require functional kinetochores (ref.31; herein incorporated by reference in its entirety). These data demonstrate that the interaction between Mps1 and the phosphodomain of Spc105 is both necessary and sufficient to activate the SAC. IT is contemplated that the kinetochore serve as the scaffold that makes this interaction sensitive to microtubule attachment; although, the present invention is not limited to any particular mechanism of action and an understanding of the mechanism of action is not necessary to practice the present invention.

Spc105120:329 Activates the SAC when Anchored in the Outer Kinetochore, but not the Inner Kinetochore

Data herein indicate an organization of Mps1 and Spc105 relative to one another that makes their interaction sensitive to the attachment state of the kinetochore. When Mps1 is anchored in the inner kinetochore, proximal to the phosphodomain of Spc105, it activates the SAC constitutively even from attached kinetochores. In contrast, if it is anchored in the outer kinetochore, distal from the phosphodomain of Spc105, it activates the SAC conditionally, only from unattached kinetochores (FIG. 11 ). Therefore, to implement attachment-sensitive SAC signaling, endogenous Mps1 bind to a site within the outer kinetochore. Consistent with this, Mps1 physically interacts with the CH domain of Ndc80, which is located in the outer kinetochore (ref. 32, 33; herein incorporated by reference in their entireties).

To investigate whether endogenous Mps1 binds within the outer kinetochore, Spc105120: 329 at N-Ndc80 was anchored proximal to the CH domain (FIG. 9 d , top). In metaphase cells, the anchored Spc105120: 329 exhibited the stereotypical, metaphase kinetochore distribution: two distinct puncta separated by <1 μm. It also recruited Mad1, and the cells remained arrested for a prolonged period (FIG. 9 d, e ). The cell cycle arrest was absent when Spc105120: 329:6A was anchored to N-Ndc80, revealing that the phosphorylation of the MELT motifs in Spc105120: 329 by kinetochore-localized Mps1 is required for SAC activation. These results demonstrate that catalytically active Mps1 binds to the outer kinetochore even after stable microtubule attachments form.

The entire kinetochore was then probed for additional Mps1-binding sites (FIG. 12 a ). When Spc105120: 329 was anchored to Dam1 subunits expected to face towards the outer kinetochore (Ask1-C, Dam1-C, or Dad1-C, see FIG. 7 f ), the kinetochores recruited Mad1, and the cells arrested in mitosis (FIG. 12 b top 12c). Strikingly, when Spc105120: 329 was anchored to positions in the inner kinetochore, including the Dam1 subunit termini predicted to face towards the centromere (Dad4-C, Spc34-C and Spc19-C), it had no effect on the cell cycle (FIG. 12 b bottom and 12c). Spc105120: 329:6A did not affect the cell cycle when anchored at any of the positions (FIG. 12 b , dashed lines). These results demonstrate that catalytically active Mps1 is absent from the inner kinetochore.

The N terminus of Spc105 localizes to the inner kinetochore and contains a Glc7-binding motif (ref. 18; herein incorporated by reference in its entirety), which is not present in Spc105120: 329. Therefore, the lack of Glc7 activity in the outer kinetochore, rather than localized Mps1 activity, could also produce the observed SAC activation phenotypes. To determine whether this is the case, a phosphodomain was constructed that contains the Glc7-binding motif (Spc1052: 329, FIG. 12 d ). Spc1052: 329 anchored at N-Ndc80 or at Ndc80-C produced the same phenotypes as Spc105120: 329 (FIG. 12 d , top). To determine whether Spc1052: 329 recruits Glc7 activity, either Spc1052: 329 or Spc105120: 329 was anchored to Ask1-C, and the kinetochore-recruitment of Bub3 was quantified (FIG. 12 d ). As Bub3 specifically binds to phosphorylated MELT motifs (ref 34; herein incorporated by reference in its entirety), significantly reduced Bub3 recruitment in the former case confirmed that Spc1052: 329 recruits Glc7 activity (FIG. 12 d , bottom). These data build an activity map for Spc105120: 329 and demonstrate that catalytically active Mps1 kinase binds exclusively in the outer kinetochore even after the kinetochore establishes stable microtubule attachment. Strikingly, this map is the mirror image of the activity map for the anchored Mps1 kinase, with the Dam1 complex demarcating the boundary in both maps (FIGS. 7 f and 12 e ). These data indicate that the Dam1 complex contributes to SAC silencing by acting as a physical barrier that separates the phosphodomain of Spc105 from Mps1.

Separation Between CH Domains of Ndc80 and N-Spc105 Changes with the Attachment State of the Kinetochore

Data indicates that microtubule attachment to kinetochores physically separates the CH domains of Ndc80 and the phosphodomain of Spc105 to silence the SAC. By corollary, unattached kinetochores bring them in close proximity to activate the SAC. To determine whether the separation between these two domains and the attachment state of the kinetochore are correlated, FRET was measured between N-Spc105 and either N-Nuf2 or N-Ndc80, which are proximal to the CH domains (FIGS. 13 a and 14). In both cases, FRET was undetectable in metaphase as predicted by the >30 nm separation between N-Spc105 and both N-Ndc80 and N-Nuf2 (ref. 20; herein incorporated by reference in its entirety)). In contrast, moderate FRET was detected in unattached kinetochores created by treating the cells with nocodazole, indicating that mCherry and GFP fused to the respective N termini were, on average, ˜8 nm apart35.

Proximity Between the CH Domains and Spc105120:329 Controls SAC Signalling in Attached Kinetochores Independently of the Endogenous Spc105

Experiments were conducted during development of embodiments herein to investigate whether Spc105120: 329 restores the SAC in attached and unattached kinetochores in a position-dependent manner in spc105-6A strains that are SAC-deficient. The kinetochore provides only the architectural scaffold in this experiment. Spc105120: 329 arrested the cell cycle when anchored proximal to the CH domains (at N-Ndc80), but not when anchored distal to the CH domains (at Spc24-C, FIG. 13 b ). Even within unattached kinetochores, Spc105120: 329 restored the SAC when it was anchored at N-Ndc80, as expected (FIG. 15 ). However, Spc105120: 329 anchored at Spc24-C also activated the SAC in unattached kinetochores, indicating that Mps1 accesses Spc105120: 329, even though its anchoring position is expected to be distal to the CH domains. It is contemplated that the inherent flexibility of Ndc80 and Spc105 and the presence of multiple molecules of these proteins in the kinetochore are responsible for this unexpected phenotype.

Example 2 Strain and Plasmid Construction

Strains used in the anchoring experiments were constructed by deleting FPR1 in wild-type strains to eliminate the rapamycin-binding protein product of this gene. These strains also express tor1-1, which encodes the dominant-negative, rapamycinresistant form of the Tor1 kinase. At least one copy of TOR1 in diploid strains was mutated to tor1-1.

Frb-GFP(S65T) (or Frb alone) was fused to the C terminus of selected SAC proteins with a 24- or 7-amino-acid linker (with the amino acid sequence ‘RIPGLINSGGGGGSGGGSGGGGAS’ (SEQ ID NO:10) or ‘SGGGGAS’ (SEQ ID NO:11), respectively). Two tandem copies of Fkbp12 (2xFkbp12) were fused to the C terminus of kinetochore proteins with the linker coding ‘RIPGLIK’ (SEQ ID NO:12). 2xFkbp12 was fused to the N terminus of Ndc80 through the linker sequence ‘GAAAAG’ (SEQ ID NO:13). A 7-amino-acid linker (sequence: ‘RIPGLIN’ (SEQ ID NO:14)) was used to fuse fluorescent proteins (either GFP(S65T) or mCherry) to the amino or carboxy terminus of selected proteins.

spc105-6A strains were constructed using plasmid shuffling. The genomic copy of SPC105 was deleted in a parent strain containing a centromeric plasmid containing SPC105 and the URA3 gene as the auxotrophic marker (pAJ274). Next, pSB1878 linearized with NsiI was integrated at the his3 locus (ref. 12; herein incorporated by reference in its entirety). Finally, the centromeric plasmid carrying the wild-type SPC105 was kicked out by counter-selecting for URA3 on the drug 5-FOA.

Plasmids containing the minimal phosphodomain of Spc105, pAJ349 and pAJ350 were constructed by subcloning the PCR amplification product of the phosphodomain of Spc105 (amino acids: 120-329 from pSB1332 for wild-type, or from pSB1878 for the phosphonull version12) into pAFS144 carrying the frb domain using AatII and KasI sites. These plasmids, after linearization with NsiI, were integrated at the his3 locus. For integration at the LEU2 locus, the HIS3 gene in pAJ349 and pAJ350 was replaced with LEU2 to construct pAJ351 and pAJ352, respectively. The plasmids were linearized with BstEII for integration at the leu2 locus.

Cell Culture

Cells were grown in yeast extract, peptone and dextrose (YPD) media at 32° C. and imaged at room temperature in synthetic media supplemented with essential amino acids and an appropriate carbon source. To express N-terminally labelled kinetochore proteins from the galactose promoter (pGAL1), strains were grown in YP Raffinose media supplemented with 0.1-0.4% galactose. The galactose concentration was adjusted empirically (ref. 35; herein incorporated by reference in its entirety).

Stock solution (1 mg ml⁻¹) of rapamycin in DMSO was diluted ×1,000 to achieve 1 μg ml⁻¹ final concentration in all experiments involving rapamycin-induced dimerization.

To depolymerize metaphase spindles with nocodazole64, mid-log-phase cells were synchronized in G1 with α-factor (2 μg ml⁻¹) for 2 h and then released into nocodazole-containing media (15 μg ml⁻¹) for 1.5-2 h.

Benomyl Sensitivity Assay

Tenfold serial dilutions of log-phase cultures were frogged on YPD or plates containing (30 μg ml-1) benomyl. Colonies were allowed to develop for 2-3 days at 30° C. before pictures of the plates were taken.

Metaphase Arrest by CDC20 Repression

Cells expressing Cdc20 from a methionine-repressible promoter (pMET3) were synchronized in G1 by treatment with α-factor (2 μg ml⁻¹) for 2 h in synthetic media lacking methionine. They were then released into YPD supplemented with 2 mM methionine for two hours to repress CDC20 and then treated with rapamycin for 10 min. Cells were washed into synthetic media lacking methionine to initiate CDC20 expression.

Inhibiting Ipl1 or Mps1 Kinase Activity Using ATP Analogues

The ATP analogues 1-NMPP1 and 1-NAPP1 (final concentration 50 μM) were used to block the activity of mps1-as1 and ipl1-as6, respectively. Cells were first synchronized in S-phase using 100 mM hydroxyl urea (HU) for 2.5 h, washed with YPD, and then released into media containing the appropriate inhibitor for 15 min. This was followed by the addition of rapamycin to the media to anchor Mps1-Frb at Mtw1-C. The bud size was used to monitor cell-cycle progress.

To examine the ability of 1-NMPP1 to block the kinase activity of mps1-as1, the cells were treated with nocodazole to depolymerize the spindle and activate the SAC (ref. 65; herein incorporated by reference in its entirety). Next, the cells were treated with either 1-NMPP1 or DMSO, and cell morphology was monitored. Mps1 kinase activity is necessary to maintain an active SAC and arrest the cells in mitosis. If the SAC remains active, then the cells remain arrested in mitosis as large-budded cells. However, SAC-deficient cells escape the mitotic arrest and also fail in cytokinesis. They enter the next cell cycle and produce another bud thus giving rise to two-budded cells (ref. 66; herein incorporated by reference in its entirety).

To study the effect of 1-NAPP1 on ipl1-as6 activity, the spindle localization of Sli15-GFP was measured in pre-anaphase cells67. The bud size was used to find pre-anaphase cells. If the bud was smaller than 50% in size as compared with the mother cell, and contained a short bar of Sli15-GFP located within the mother cell and at the bud neck, then the cell was deemed to be in pre-anaphase.

Scoring Mitotically Arrested Cells

The cells were scored as ‘large-budded’ (for example, FIGS. 1 e and 5 b ), if the size of the bud was more than ⅔ the size of the mother as seen from bright-field images. Anaphase cells in cycling cultures will also be scored as large-budded cells by this criterion. In strains carrying fluorescent markers, the separation of the kinetochore clusters or spindle pole bodies was used to determine whether or not the cells arrested in mitosis. Large-budded cells with kinetochore-cluster separation smaller than 1 μm or spindle length smaller than 2 μm were scored as metaphase-arrested cells (ref. 68; herein incorporated by reference in its entirety).

Colony-Counting Assays

Approximately 300 cells (estimated from the attenuance of liquid cultures measured at 660 nm) were plated on control and rapamycin-containing plates. After allowing the colonies to grow for 3 days at 30° C., colony number was determined. It was ensured that the strains used in this experiment were rapamycin-resistant, by verifying that the parental haploid strains expressing either the Frb-fused SAC protein or the Fkbp12-fused kinetochore protein produced the same number of colonies on both control and rapamycin-containing plates.

Microscopy and Image Acquisition

A Nikon Ti-E inverted microscope with a 1.4 NA, 100×, oil-immersion objective was used in imaging (ref. 35; herein incorporated by reference in its entirety). A ten-plane Z-stack was acquired (200 nm separation between adjacent planes). The total fluorescence from each kinetochore cluster with GFP- or mCherry-tagged protein was measured using ImageJ, or a semi-automated MATLAB program. The copy numbers of kinetochore proteins and anchored proteins were calculated from the known copy number of the Ndc80 complex per kinetochore-8 molecules per kinetochore.

For photobleaching, an argon-ion laser (Photonics Instruments) beam filtered with the ET-GFP filter cube was focused on the sample by the objective. The target was manually aligned with the pre-determined location of the laser focus, and then exposed to 488 nm light for 50 ms. Five-plane Z-stacks were acquired starting immediately after bleaching for 14 min, at 2 min intervals. Fluorescence was quantified from the images as above.

FRET, high-resolution co-localization and fluorescence distribution analyses were conducted as previously described in ref. 20, 35, and 69; herein incorporated by reference in their entireties.

Time-lapse imaging was used to follow the Mps1-Frb-GFP that autonomously bound to the kinetochore clusters in metaphase-arrested cells. Cells were released from the metaphase arrest by activating CDC20 expression, and a 6-plane Z-stack was acquired at 1 min intervals for 20 min. Anaphase entry was inferred from spindle elongation tracked from the spindle pole body protein (Spc97-mCherry). The change in Mps1-Frb-GFP intensity during this period was quantified, after correcting for two factors: GFP photobleaching expected from imaging and; fluorescence emission from Spc97-mCherry due to cross-excitation while imaging GFP. The representative images in FIG. 2 f have not been corrected for these factors.

Example 3

Experiments conducted during development of embodiments herein demonstrate control of the yeast SAC independently of the kinetochore by ‘short-circuiting’ the kinetochore-based mechanical switch (See, e.g., Example 1). Further experiments have demonstrated that this short-circuiting approach also works in human cells. Recombinant-mediated cassette exchange (RMCE) was used to stably integrate a cassette that constitutively expresses M3-M3-neonGFP-2xFkbp12, wherein M3-M3 corresponds to a fragment of the phosphodomain of human KNL1 containing 6 MELT motifs (a kind gift from the Kops lab. This protein construct did not localize to the kinetochore. Frb-mCherry-Mps1 is conditionally expressed by a TetON promoter (FIG. 16 ). Cells expressing both fragments underwent a prolonged arrest in metaphase when rapamycin was added to the culture media (FIG. 16 ). This arrest was absent in the absence of rapamycin, or even in the presence of rapamycin if the cell did not express a detectable level of Frb-Mps1.

To verify that the metaphase arrest is due to the activation of SAC, HeLa cells are synchronized using double thymidine block, released into the cell cycle, and cell-cycle progress is monitored in the presence and absence of rapamycin using biochemical markers (Pds1/Securin levels) and microscopic examination (fixed cells stained for phospho-histone 113). Analog-sensitive Mps1 and non-phosphorylatable M3-M3 allele are used to ensure that the arrest results from the phosphorylation of M3-M3 by Mps1. It is tested whether dimerization activates the SAC even when Ndc80/Hec1, which is essential for SAC signaling from the kinetochore, is knocked-down. SAC activation in the absence of Ndc80 confirms that the kinetochores do not participate in the signaling induced by the cytosolic dimerization of Mps1 and M3-M3. Together, these experiments demonstrate that the interaction between Mps1 and KNL1 is both necessary and sufficient for activating the SAC in human cells. These experiments indicate that the function of the kinetochore is to control this interaction, and make it sensitive to microtubule attachment.

Example 4

As described elsewhere herein, a kinetochore-independent SAC activator was engineered which comprises a region of KNL1 that contains a series of Mps1 phosphorylation sites, known as “MELT repeats”, which are necessary for SAC activation, but lacks the kinetochore-localization domain at the C-terminus or the newly identified transient localization domain at its N-terminus (FIG. 21 ). FKBP12 was fused to this minimal KNL1 phosphodomain and FRB to Mps1, and rapamycin was used to induce dimerization of the fusion proteins in HeLa cells (FIGS. 17B, 22 ). Upon induction of KNL1 phosphodomain-Mps1 dimerization by adding rapamycin, cells displayed a prolonged mitotic arrest indicative of SAC activation (FIG. 17C). These cells maintained an aligned metaphase plate indicating an inability to initiate anaphase and that the observed mitotic arrest was not due to the disruption of kinetochore-microtubule interactions. The arrest was reversed rapidly upon inhibition of Mps1 kinase activity by the small molecule inhibitor Reversine, indicating that it required Mps1 kinase activity (FIG. 17D). To further ensure kinetochore-independent operation of the dimerized proteins, the kinetochore-localization domain of Mps1 was removed (FIG. 17E). Rapamycin-induced dimerization of this minimal Mps1 kinase domain with the minimal KNL1 phosphodomain also produced a sustained mitotic arrest without any detectable kinetochore localization (FIG. 17E). Furthermore, individually inhibiting the kinase activity of exogenous Mps1 using an analog-sensitive version of the kinase domain or preventing the phosphorylation of the minimal KNL1 phosphodomain by using a non-phosphorylatable version of the phosphodomain each prevented the rapamycin-induced mitotic arrest (FIG. 23 ). Thus, the catalytic activity of the Mps1 kinase domain and the Mps1 phosphorylation sites within the minimal phosphodomain are both essential for the rapamycin-induced metaphase arrest. These data demonstrate that phosphorylation of the MELT repeats in KNL1 by Mps1 in the cytosol is sufficient to institute a metaphase arrest in human cells. The above described system of FKBP12/FRB-dimerizable Mps1 kinase domain and KNL1 phosophodomains is referred to in this example as an ectopic SAC activation system (eSAC).

Experiments were conducted during development of embodiments herein to demonstrate that the eSAC is completely independent of the kinetochore-based SAC activation machinery. MELT repeats in the eSAC phosphodomain were phosphorylated only in the presence of rapamycin, and were not phosphorylated if the SAC was activated by creating unattached kinetochores by treatment with the microtubule depolymerizing drug nocodazole (FIG. 18A). Reciprocally, the MELT repeats in endogenous KNL1 were not appreciably phosphorylated in rapamycin-treated cells, but were strongly phosphorylated in nocodazole-treated cells. Thus, there is negligible cross-talk in the phosphoregulation of the eSAC phosphodomain and endogenous KNL1. The kinetochore-based SAC signaling was selectively inactivated by Reversine treatment (FIG. 18B, left) to determine whether an eSAC, which uses a partially Reversine-resistant allele of the Mps1 kinase domain, still arrests mitosis (FIG. 18B). Activation of this eSAC significantly delayed mitosis even in the presence of Reversine, demonstrating that kinetochore-based SAC signaling is dispensable for eSAC activity (FIG. 18B, right). The observed reduction in the average mitotic time was expected because the Mps1 allele is only partially resistant to Reversine (IC50 ˜130 nM compared to ˜30 nM for the wild-type Mps1). Kinetochore-independent operation of the eSAC was further confirmed by the observation that inhibiting Aurora B kinase activity, which contributes to the recruitment of Mps1 and SAC proteins to kinetochores, did not reduce the eSAC-induced mitotic delay (FIG. 18C). Next, the eSAC phosphodomain was targeted to the plasma membrane by incorporating a palmitoylation sequence at its N-terminus; when complexed with the Mps1 kinase domain, this membrane-tethered phosphodomain induced a potent mitotic arrest similar to that induced by its cytosolic version (FIG. 18D). The experiments conducted during development of embodiments herein demonstrate that the eSAC operates independently of the kinetochore to inhibit anaphase.

Experiments were conducted during development of embodiments herein to define the events that occur downstream of eSAC activation. Mass spectrometry analysis was performed on immunoprecipitated eSAC phosphodomain (FIG. 18E). When the eSAC phosphodomain was isolated from cells arrested in mitosis by nocodazole, this analysis identified peptides from KNL1 and FKBP12, but not from Mps1 or any of the SAC proteins. In contrast, affinity purification of the eSAC phosphodomain following rapamycin treatment isolated the SAC proteins Bub3 and Bub1, in addition to the Mps1 kinase domain. To identify dynamic interacting partners for the eSAC phosphodomain, cells were treated with the crosslinking agent formaldehyde prior to the affinity purification to trap weakly associated proteins. These purifications additionally isolated BubR1, a key component of the Mitotic Checkpoint Complex (MCC). Thus, the eSAC phosphodomain recruits components of the SAC signaling cascade and the mitotic checkpoint complex only when it is phosphorylated by Mps1. Next, the eSAC was activate in cells depleted for BubR1 or Mad2, the essential components of the MCC, using RNAi, to determine whether the eSAC-induced metaphase arrest requires the formation of the MCC. In both cases, rapamycin-treatment was unable to cause a mitotic arrest (FIG. 18F). The data indicate that eSAC activation generates phosphorylated MELT repeats in the cytosol, which then recruit SAC proteins and catalyze the formation of the MCC. The MCC inhibits Anaphase Promoting Complex, and delays anaphase onset. Thus, the eSAC is a minimal, but potent, system that institutes a controllable biochemical block to anaphase without interfering with the mechanics of cell division.

Experiments conducted during development of embodiments herein to analyze the dose-response characteristics of the eSAC: the relationship between the abundance of the dimeric eSAC activator and the corresponding duration of mitotic arrest (FIG. 19B-C). In these assays, it was found that the duration of mitosis was strongly affected only by the limiting abundance of the mCherry-tagged Mps1 kinase domain, and not by the abundance of the highly-expressed phosphodomain (FIG. 24 ). Therefore, mCherry fluorescence intensity was used as the measure of the eSAC activator complex (FIG. 24 ).Frb-mCherry-Mps1 abundance was in a range comparable to that of endogenous Mps1 (FIG. 25 ). Therefore, the dose-response curves probed SAC function in a physiologically relevant concentration range.

It was found that the cellular abundance of the eSAC activator and the number of MELT repeats per eSAC phosphodomain had striking, systematic effects on the duration of mitosis. With phosphodomains containing up to four MELT repeats, each eSAC dose-response relationship was sigmoidal (FIGS. 19 -E, 26). Each curve possessed a characteristic ‘activation threshold’, defined as the eSAC activator abundance necessary to increase mitotic duration by 10% over its baseline value (FIG. 19D-E). Beyond this threshold, mitotic duration increased proportionally with eSAC activator abundance before reaching a plateau. As the number of MELT repeats in the eSAC phosphodomains increased, the activation threshold decreased, the slope of the linear regime increased. The final, asymptotic delay in anaphase onset also increased, but in a complex, non-intuitive manner.

Because the eSAC delays mitosis by stimulating the SAC signaling cascade, its dose-response characteristics reflect the relationship between the steady-state concentration of MCC and the duration of mitosis. Thus, beyond their respective activation thresholds, eSAC phosphodomains containing up to 4 MELT repeats generate gradually increasing concentrations of MCC. Consequently, over this limited range of eSAC activator abundance, the operation of the SAC signaling cascade resembles that of a rheostat resisting anaphase onset. Saturation of the maximal time in mitosis at high eSAC concentrations indicates that MCC generation does not increase any further, for example, because of the limited concentration or activity of downstream SAC proteins. The activation threshold and the steepness of the dose-response curve are both critical characteristics of each phosphodomain, because they indicate the smallest concentration of the respective phosphodomain that delays anaphase onset and the signaling strength per molecule respectively. Increasing the number of MELT repeats per eSAC phosphodomain reduced the activation threshold and increased the signaling strength approximately proportionally (FIG. 19G-I). This trend partially explains why multiple MELT repeats per KNL1 are evolutionarily favored.

The data imply that endogenous KNL1 will possess a much smaller activation threshold and higher signaling strength, because it contains 19 MELT repeats. However, KNL1 alleles with only 6 MELT repeats are capable of recruiting the same number of Bub3-Bub1 molecules and activating the SAC as wild-type KNL1. This counter-intuitive finding is explained by the complex dose-response relationship for the eSAC phosphodomain with 6 MELT repeats (FIG. 19F). This phosphodomain displayed a surprisingly low activation threshold and high signaling strength. It also achieved a disproportionately large increase in the maximal duration of mitosis (FIG. 19G-I). In effect, the phosphodomain containing 6 MELT repeats stimulated the SAC signaling cascade like a switch (FIGS. 19F, 27 ). The disproportionately large increase in signaling strength suggests that the concurrent recruitment of SAC proteins by MELT repeats within the same phosphodomain molecule produces a synergistic output. This conclusion is further bolstered by the gradual decline in the response with increasing concentrations of the eSAC activator. As the eSAC activator concentration increases, the phosphodomains compete with one-another to recruit the limited pool of downstream signaling proteins. Consequently, individual eSAC phosphodomains no longer recruit multiple SAC proteins, which diminishes synergistic activity. Therefore, at high eSAC concentration the dose-response curve for the phosphodomain with 6 MELT repeats approaches the asymptotic plateau for eSAC phosphodomains containing 3-4 repeats (FIG. 19G).

A mathematical model of the eSAC (FIG. 20 ) was constructed which assumes that each MELT motif recruits SAC proteins with a characteristic affinity (Table 1). The model represents all the SAC proteins are represented by a single factor named ‘Bub’, because quantitative measurements for these recruitment reactions are not available (FIG. 20A-B). For eSAC phosphodomains containing more than one MELT motif, the model calculates the steady state concentration of all possible species of the phosphodomain characterized by MELT motifs bound by the SAC proteins (concentrations for eSAC phosphodomains with one and 4 MELT repeats displayed in the middle graphs in FIGS. 20A and B, respectively). The model further assumes that the abundance of one or more SAC proteins is lower than the abundance of the eSAC activator. Consequently, the abundance of eSAC phosphodomains bound with different numbers of SAC proteins strongly depends on the eSAC activator abundance (FIGS. 20A-B, 28). The steady-state MCC generated by kinetochores depends on the amount of SAC proteins that they recruit. Therefore, it was assume that the rate of conversion of the SAC-active form of Mad2 (FIG. 20A-B, right panels), and hence the steady-state MCC concentration generated by the eSAC, is proportional to the number of SAC proteins recruited by the eSAC phosphodomains. To simulate the time in mitosis as a function of eSAC abundance, the cumulative MCC generated by all the phosphodomains was relayed to a mathematical representation of a bi-stable switch that controls the onset of anaphase (FIGS. 29-30 ). This model captured the average dose-response characteristics of eSAC phosphodomains containing up to 4 MELT repeats (FIG. 20C). However, this simple scheme did not reproduce the complex dose-response relationship for the phosphodomain with 6 MELT repeats (FIG. 20C, dashed black curve). In this case, it was assumed that eSAC phosphodomains that had more than one MELT repeat bound by SAC proteins produced MCC at a modestly higher rate (≤20% increase due to synergistic output, Table 2). With this modification, the model accurately captured the dose-response characteristics for the eSAC phosphodomain containing 6 MELT repeats (FIG. 20C).

TABLE 1 Rate constants for Bub binding. Vleugel et al. Parameter Value Parameter Value classification k_(f11) 1 nM⁻¹ min⁻¹ k_(r11) 0.1 min⁻¹ High k_(f12) 1 nM⁻¹ min⁻¹ k_(r12) 0.1 min⁻¹ High k_(f13) 1 nM⁻¹ min⁻¹ k_(r13)   5 min⁻¹ Low k_(f14) 1 nM⁻¹ min⁻¹ k_(r14) 0.1 min⁻¹ Intermediate

TABLE 2 Rate of open-Mad2 to closed/active-Mad2 conversion by the Bub binding states. All the rates were multiplied by a scaling factor 0.013 in order to scale the simulation results to match experimental data. For the sake of brevity, the MELT numbering notation has been shortened using the rule: 11->1, 12->2, 13->3, 14->4. Only the numbers of Bub-bound MELT motifs are listed. In the case of eSAC phosphodomain with 6 MELT repeats the underscore symbol is used to differentiate between the two sets of MELT 12, 13, 14. Cooperative interactions are bold outlined. Parameter Value Parameter Value Parameter Value k₄ 1.02 k₂_2   2 * k₂ k₃₄_34   2 * k₃₄ k₃ 1 k₂₃₄  k_(a) + k₃ + k₄ k₃₄_24  k₃₄ + k₂₄ k₂ 1 k₄_34  k₄ + k₃₄ k₃₄_23  k₃₄ + k₂₃ k₁ 1.01 k₄_24  k₄ + k₂₄ k₂_234  k₂ + k₂₃₄ k₃₄ k₃ + k₄ k₄_23  k₄ + k₂₃ k₂₄_34  k₂₄ + k₃₄ k₂₄ k₂ + k₄ k₃_34  k₃ + k₃₄ k₂₄_24

k₂₃ k₂ + k₃ k₃_24  k₃ + k₂₄ k₂₄_23  k₂₄ + k₂₃ k₁₄ k₁ + k₄ k₃_23  k₃ + k₂₃ k₂₃_34  k₂₃ + k₃₄ k₁₃ k₁ + k₃ k₃₄_4 k₃₄ + k₄ k₂₃_24  k₂₃ + k₂₄ k₁₂ k₁ + k₂ k₃₄_3 k₃₄ + k₃ k₂₃_23   2 * k₂₃ k₁₂₃ k₁ + k₂ + k₃ k₃₄_2 k₃₄ + k₂ k₂₃₄_4 k₂₃₄ + k₄ k₁₂₄ k₁ + k₂ + k₄ k₂_34  k₂ + k₃₄ k₂₃₄_3 k₂₃₄ + k₃ k₁₃₄ k₁ + k₃ + k₄ k₂_24  k₂ + k₂₄ k₂₃₄_2 k₂₃₄ + k₂ k₁₂₃₄ k₁ + k₂ + k₃ + k₄ k₂_23  k₂ + k₂₃ k₃₄_234  k₃₄ + k₂₃₄ k₄_4

k₂₄_4 k₂₄ + k₄ k₂₄_234

k₄_3 k₄ + k₃ k₂₄_3 k₂₄ + k₃ k₂₃_234  k₂₃ + k₂₃₄ k₄_2 k₄ + k₂ k₂₄_2 k₂₄ + k₂ k₂₃₄_34 k₂₃₄ + k₃₄ k₃_4 k₃ + k₄ k₂₃_4 k₂₃ + k₄ k₂₃₄_24

k₃_3  2 * k₃ k₂₃_3 k₂₃ + k₃ k₂₃₄_23 k₂₃₄ + k₂₃ k₃_2 k₃ + k₂ k₂₃_2 k₂₃ + k₂ k₂₃₄_234

k₂_4 k₂ + k₄ k₄_234  k₄ + k₂₃₄ k₂_3 k₂ + k₃ k₃_234  k₃ + k₂₃₄

All publications and patents provided herein incorporated by reference in their entireties. Various modifications and variations of the described compositions and methods of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the relevant fields are intended to be within the scope of the present invention.

REFERENCES

The following references, some of which are cited above by number, are herein incorporated by reference in their entireties.

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1-15. (canceled)
 16. A method of activating a spindle assembly checkpoint (SAC) in a cell comprising administering to the cell a system of claim
 20. 17. The method of claim 16, wherein activating the SAC prevents aneuploidies in the cell.
 18. The method of claim 16, wherein the cell is within a tissue, organ, or subject, and the SAC is activated in all or a group of cells within the tissue, organ, or subject.
 19. The method of claim 18, wherein activating the SAC treats or prevents cancer in the cells, tissue, organ, or subject.
 20. A cellular or in vitro biochemical system comprising: (a) a polypeptide having at 95% sequence identity to a kinase domain of the wild-type Mps1 (SEQ ID NO:2) linked to a first dimerization element; and (b) a polypeptide having at 95% sequence identity to a phosphodomain of the wild-type KNL1 (SEQ ID NO:5) linked to a second dimerization element, wherein the kinase domain is capable of phosphorylating the phosphodomain, and wherein dimerization of the first dimerization element and second dimerization element facilitates phosphorylation of the KNL1 polypeptide by the Mps1 polypeptide.
 21. The system of claim 20, wherein the phosphorylation of the phosphodomain by the kinase domain is sufficient to activate a spindle assembly checkpoint (SAC) in a cell within which the phosphorylation occurs.
 22. The system of claim 20, wherein the first or second dimerization element is Frb and the other dimerization element is Fkbp12.
 23. The system of claim 20, further comprising a dimerization inducer, wherein the dimerization inducer alters the degree of dimerization in a concentration dependent manner. 